Ex vivo MR volumetry of human brain hemispheres.

PubMed

Kotrotsou, Aikaterini; Bennett, David A; Schneider, Julie A; Dawe, Robert J; Golak, Tom; Leurgans, Sue E; Yu, Lei; Arfanakis, Konstantinos

2014-01-01

The aims of this work were to (a) develop an approach for ex vivo MR volumetry of human brain hemispheres that does not contaminate the results of histopathological examination, (b) longitudinally assess regional brain volumes postmortem, and (c) investigate the relationship between MR volumetric measurements performed in vivo and ex vivo. An approach for ex vivo MR volumetry of human brain hemispheres was developed. Five hemispheres from elderly subjects were imaged ex vivo longitudinally. All datasets were segmented. The longitudinal behavior of volumes measured ex vivo was assessed. The relationship between in vivo and ex vivo volumetric measurements was investigated in seven elderly subjects imaged both antemortem and postmortem. This approach for ex vivo MR volumetry did not contaminate the results of histopathological examination. For a period of 6 months postmortem, within-subject volume variation across time points was substantially smaller than intersubject volume variation. A close linear correspondence was detected between in vivo and ex vivo volumetric measurements. Regional brain volumes measured with this approach for ex vivo MR volumetry remain relatively unchanged for a period of 6 months postmortem. Furthermore, the linear relationship between in vivo and ex vivo MR volumetric measurements suggests that this approach captures information linked to antemortem macrostructural brain characteristics. Copyright © 2013 Wiley Periodicals, Inc.

Ex-vivo MR Volumetry of Human Brain Hemispheres

PubMed Central

Kotrotsou, Aikaterini; Bennett, David A.; Schneider, Julie A.; Dawe, Robert J.; Golak, Tom; Leurgans, Sue E.; Yu, Lei; Arfanakis, Konstantinos

2013-01-01

Purpose The aims of this work were to: a) develop an approach for ex-vivo MR volumetry of human brain hemispheres that does not contaminate the results of histopathological examination, b) longitudinally assess regional brain volumes postmortem, and c) investigate the relationship between MR volumetric measurements performed in-vivo and ex-vivo. Methods An approach for ex-vivo MR volumetry of human brain hemispheres was developed. Five hemispheres from elderly subjects were imaged ex-vivo longitudinally. All datasets were segmented. The longitudinal behavior of volumes measured ex-vivo was assessed. The relationship between in-vivo and ex-vivo volumetric measurements was investigated in seven elderly subjects imaged both ante-mortem and postmortem. Results The presented approach for ex-vivo MR volumetry did not contaminate the results of histopathological examination. For a period of 6 months postmortem, within-subject volume variation across time points was substantially smaller than inter-subject volume variation. A close linear correspondence was detected between in-vivo and ex-vivo volumetric measurements. Conclusion Regional brain volumes measured with the presented approach for ex-vivo MR volumetry remain relatively unchanged for a period of 6 months postmortem. Furthermore, the linear relationship between in-vivo and ex-vivo MR volumetric measurements suggests that the presented approach captures information linked to ante-mortem macrostructural brain characteristics. PMID:23440751

Ex vivo Skin Permeation of Betulin from Water-in-Oil Foams.

PubMed

Färber, Anna; Daniels, Rolf

2016-01-01

Triterpenes of the outer bark of birch are known to improve wound healing. An oleogel with these triterpenes as active principle is approved by the European Medicines Agency. As foams can be applied without touching the skin, they might be an advantageous application form. A comparable wound-healing effect can be expected when the permeation flux of the triterpenes from different types of formulations, namely oleogels, water-in-oil emulsions and water-in-oil foams, is similar. The tested formulations were based on three lipids (medium-chain trigylcerides, sunflower oil and paraffin) which differ in their polarity and solvent power for the triterpenes. Infinite dose permeation experiments were performed using porcine skin which was injured by either tape stripping or skin grafting. The results showed that steady-state permeation flux and lag time depend clearly on the depth of the skin lesion. Moreover, it was substantially affected by the lipid used as basis for the different formulations. In contrast, the different formulation types showed a comparable permeation behaviour leading to the conclusion that all formulation types can be used alike for the treatment of wounds, and the results that have already been obtained with oleogels can be directly translated to the foam with its superior use properties. © 2016 S. Karger AG, Basel.

Transbuccal delivery of betahistine dihydrochloride from mucoadhesive tablets with a unidirectional drug flow: in vitro, ex vivo and in vivo evaluation

PubMed Central

El-Nabarawi, Mohamed A; Ali, Adel A; Aboud, Heba M; Hassan, Amira H; Godah, Amany H

2016-01-01

Objective Betahistine dihydrochloride (BH.2HCl), an anti-vertigo histamine analog used in the treatment of Ménière’s disease, undergoes extensive first-pass metabolism and suffers from short biological half-life. The aim of the present work was to develop and estimate controlled release mucoadhesive buccal tablets of BH.2HCl with a unidirectional drug flow to overcome this encumbrance. Methods A direct compression method was adopted for preparation of the tablets using mucoadhesive polymers like guar gum, hydroxypropyl methyl cellulose K4M, sodium carboxymethyl cellulose and their combinations. The tablets were coated from all surfaces except one surface with a solution of 5% (w/v) cellulose acetate and 1% (w/v) dibutyl phthalate. Different permeation enhancers like 2% sodium deoxycholate, 2% sodium cholate hydrate (SCH) and 5% menthol were tested. Swelling index, ex vivo residence time, mucoadhesion strength, in vivo testing of mucoadhesion time, in vitro dissolution and ex vivo permeation were carried out. Furthermore, compatibility and accelerated stability studies were performed for the drug excipients. Finally, drug bioavailability of the BH.2HCl-optimized buccal mucoadhesive formulation was compared with that of the orally administered Betaserc® 24 mg tablet in six healthy male volunteers. Results Formulation F10, which contained a combination of 35% guar gum and 5% sodium carboxymethyl cellulose, exhibited long adhesion time, high adhesion strength and diminished irritation to volunteers and showed zero-order release kinetics. SCH produced a significant enhancement in permeation of BH.2HCl across buccal mucosa. BH.2HCl-optimized buccal mucoadhesive formulation showed percentage relative bioavailability of 177%. Conclusion The developed mucoadhesive tablets represent a promising alternative for the buccal delivery of BH.2HCl. PMID:28008227

Ex vivo lung perfusion.

PubMed

Reeb, Jeremie; Cypel, Marcelo

2016-03-01

Lung transplantation is an established life-saving therapy for patients with end-stage lung disease. Unfortunately, greater success in lung transplantation is hindered by a shortage of lung donors and the relatively poor early-, mid-, and long-term outcomes associated with severe primary graft dysfunction. Ex vivo lung perfusion has emerged as a modern preservation technique that allows for a more accurate lung assessment and improvement in lung quality. This review outlines the: (i) rationale behind the method; (ii) techniques and protocols; (iii) Toronto ex vivo lung perfusion method; (iv) devices available; and (v) clinical experience worldwide. We also highlight the potential of ex vivo lung perfusion in leading a new era of lung preservation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ex-Vivo percutaneous absorption of enrofloxacin: Comparison of LMOG organogel vs. pentravan cream.

PubMed

Kirilov, Plamen; Tran, Van Hung; Ducrotté-Tassel, Alban; Salvi, Jean-Paul; Perrot, Sébastien; Haftek, Marek; Boulieu, Roselyne; Pirot, Fabrice

2016-02-10

The objective of this study was to investigate the percutaneous absorption of enrofloxacin from two base formulations, Pentravan cream and LMOG organogel. Ex-vivo experiments were carried out on pig ear skin. The percutaneous permeation through pig skin of two formulations containing 5 wt% of enrofloxacin was measured and compared using Franz diffusion cells. At appropriate intervals up to 120 h, diffusion samples were taken and analyzed using HPLC assays. Permeation profiles were established and the parameters Tlag and flux values were calculated. In this ex-vivo study, the flux values were 0.35 μgcm(-2)h(-1) for Pentravan and 1.22 μgcm(-2)h(-1) for LMOG organogel, corresponding respectively to 7.9 % and 29.3 % of enrofloxacin absorbed after 120 h by these formulations. The lag time (T lag) of Pentravan and organogel were 6.32 and 0.015 h respectively. The absorption time to reach the antibiotic concentration of enrofloxacin (2 μgmL(-1)) in the receptor was 60 h with Pentravan and 30 h with the organogel, suggesting more effective treatment by the latter. Enrofloxacin contained in organogel could be absorbed through pig ear skin 3.7 times greater than that in Pentravan (commercial formulation). This study demonstrates the perspective of organogel formulations as potential drug delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Tolterodine Tartrate Proniosomal Gel Transdermal Delivery for Overactive Bladder

PubMed Central

Rajabalaya, Rajan; Leen, Guok; Chellian, Jestin; Chakravarthi, Srikumar; David, Sheba R.

2016-01-01

The goal of this study was to formulate and evaluate side effects of transdermal delivery of proniosomal gel compared to oral tolterodine tartrate (TT) for the treatment of overactive bladder (OAB). Proniosomal gels are surfactants, lipids and soy lecithin, prepared by coacervation phase separation. Formulations were analyzed for drug entrapment efficiency (EE), vesicle size, surface morphology, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, in vitro skin permeation, and in vivo effects. The EE was 44.87%–91.68% and vesicle size was 253–845 nm for Span formulations and morphology showed a loose structure. The stability and skin irritancy test were also carried out for the optimized formulations. Span formulations with cholesterol-containing formulation S1 and glyceryl distearate as well as lecithin containing S3 formulation showed higher cumulative percent of permeation such as 42% and 35%, respectively. In the in vivo salivary secretion model, S1 proniosomal gel had faster recovery, less cholinergic side effect on the salivary gland compared with that of oral TT. Histologically, bladder of rats treated with the proniosomal gel formulation S1 showed morphological improvements greater than those treated with S3. This study demonstrates the potential of proniosomal vesicles for transdermal delivery of TT to treat OAB. PMID:27589789

Ex-vivo quantitative susceptibility mapping of human brain hemispheres

PubMed Central

Kotrotsou, Aikaterini; Tamhane, Ashish A.; Dawe, Robert J.; Kapasi, Alifiya; Leurgans, Sue E.; Schneider, Julie A.; Bennett, David A.; Arfanakis, Konstantinos

2017-01-01

Ex-vivo brain quantitative susceptibility mapping (QSM) allows investigation of brain characteristics at essentially the same point in time as histopathologic examination, and therefore has the potential to become an important tool for determining the role of QSM as a diagnostic and monitoring tool of age-related neuropathologies. In order to be able to translate the ex-vivo QSM findings to in-vivo, it is crucial to understand the effects of death and chemical fixation on brain magnetic susceptibility measurements collected ex-vivo. Thus, the objective of this work was twofold: a) to assess the behavior of magnetic susceptibility in both gray and white matter of human brain hemispheres as a function of time postmortem, and b) to establish the relationship between in-vivo and ex-vivo gray matter susceptibility measurements on the same hemispheres. Five brain hemispheres from community-dwelling older adults were imaged ex-vivo with QSM on a weekly basis for six weeks postmortem, and the longitudinal behavior of ex-vivo magnetic susceptibility in both gray and white matter was assessed. The relationship between in-vivo and ex-vivo gray matter susceptibility measurements was investigated using QSM data from eleven older adults imaged both antemortem and postmortem. No systematic change in ex-vivo magnetic susceptibility of gray or white matter was observed over time postmortem. Additionally, it was demonstrated that, gray matter magnetic susceptibility measured ex-vivo may be well modeled as a linear function of susceptibility measured in-vivo. In conclusion, magnetic susceptibility in gray and white matter measured ex-vivo with QSM does not systematically change in the first six weeks after death. This information is important for future cross-sectional ex-vivo QSM studies of hemispheres imaged at different postmortem intervals. Furthermore, the linear relationship between in-vivo and ex-vivo gray matter magnetic susceptibility suggests that ex-vivo QSM captures

Lipid drug conjugate nanoparticle as a potential nanocarrier for the oral delivery of pemetrexed diacid: Formulation design, characterization, ex vivo, and in vivo assessment.

PubMed

Soni, Kriti; Mujtaba, Ali; Kohli, Kanchan

2017-10-01

The present work was to develop lipid drug conjugated (LDC) nanoparticles for the potential oral delivery of pemetrexed diacid (PTX) and evaluation of its in vitro, ex vivo and in vivo potentials. The LDC was prepared by salt formation of PTX with stearic acid and followed by cold homogenization technique to produce the LDC nanoparticles. FTIR analysis of LDC proved the presence of amide bond in LDC powder indicating the conjugation between drug and lipid. LDC nanoparticles was found to have particle size 121.9±1.85nm and zeta potential -51.6mV±1.23 and entrapment efficiency 81.0±0.89%. TEM images revealed spherical morphology and were in corroboration with particle size measurements. Ex vivo gut permeation studies revealed a very good enhancement in permeation of drug present in the LDC as compared to plain drug solution and were confirmed by CLSM. MTT assay conformed significant% toxicity at the end of 24h and 48h. Furthermore, the AUC 0-24 of PTX from the optimized LDC nanoparticels was found to be 4.22 folds higher than that from PTX suspension on oral administration. Thus, LDC has high potential for the oral delivery of PTX in cancer therapy and future prospects for the industrial purpose. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a convenient ex vivo model for the study of the transcorneal permeation of drugs: histological and permeability evaluation.

PubMed

Pescina, Silvia; Govoni, Paolo; Potenza, Arianna; Padula, Cristina; Santi, Patrizia; Nicoli, Sara

2015-01-01

In this paper, an ex vivo model for the study of the transcorneal permeation of drugs, based on porcine tissues, was evaluated. The setup is characterized by ease of realization, absence of O₂ and CO₂ bubbling and low cost; additionally, the large availability of porcine tissue permits a high throughput. Histological images showed the comparability between porcine and human corneas and confirmed the effectiveness of the isolation procedure. A new de-epithelization procedure based on a thermal approach was also set up to simulate cornea permeability in pathological conditions. The procedure did not affect the integrity of the underlying layers and allowed the characterization of the barrier properties of epithelium and stroma. Six compounds with different physicochemical properties were tested: fluorescein, atenolol, propranolol, diclofenac, ganciclovir and lidocaine. The model highlighted the barrier function played by epithelium toward the diffusion of hydrophilic compounds and the permselectivity with regard to more lipophilic molecules. In particular, positively charged compounds showed a significantly higher transcorneal permeability than negatively charged compounds. The comparability of results with literature data supports the goodness and the robustness of the model, especially taking into account the behavior of fluorescein, which is generally considered a marker of tissue integrity. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Ex Vivo ERG analysis of photoreceptors using an In Vivo ERG system

PubMed Central

Vinberg, Frans; Kolesnikov, Alexander V.; Kefalov, Vladimir J.

2014-01-01

The Function of the retina and effects of drugs on it can be assessed by recording transretinal voltage across isolated retina that is perfused with physiological medium. However, building ex vivo ERG apparatus requires substantial amount of time, resources and expertise. Here we adapted a commercial in vivo ERG system for transretinal ERG recordings from rod and cone photoreceptors and compared rod and cone signalling between ex vivo and in vivo environments. We found that the rod and cone a- and b-waves recorded with the transretinal ERG adapter and a standard in vivo ERG system are comparable to those obtained from live anesthetized animals. However, ex vivo responses are somewhat slower and their oscillatory potentials are suppressed as compared to those recorded in vivo. We found that rod amplification constant (A) was comparable between ex vivo and in vivo conditions, ∼10 - 30 s-2 depending on the choice of response normalization. We estimate that the A in cones is between 3 and 6 s-2 in ex vivo conditions and by assuming equal A in vivo we arrive to light funnelling factor of 3 for cones in the mouse retina. The ex vivo ERG adapter provides a simple and affordable alternative to designing a custom-built transretinal recordings setup for the study of photoreceptors. Our results provide a roadmap to the rigorous quantitative analysis of rod and cone responses made possible with such a system. PMID:24959652

Biopharmaceutical evaluation of surface active ophthalmic excipients using in vitro and ex vivo corneal models.

PubMed

Juretić, Marina; Cetina-Čižmek, Biserka; Filipović-Grčić, Jelena; Hafner, Anita; Lovrić, Jasmina; Pepić, Ivan

2018-07-30

The objective of this study was to systematically investigate the effects of surface active ophthalmic excipients on the corneal permeation of ophthalmic drugs using in vitro (HCE-T cell-based model) and ex vivo (freshly excised porcine cornea) models. The permeation of four ophthalmic drugs (i.e., timolol maleate, chloramphenicol, diclofenac sodium and dexamethasone) across in vitro and ex vivo corneal models was evaluated in the absence and presence of four commonly used surface active ophthalmic excipients (i.e., Polysorbate 80, Tyloxapol, Cremophor® EL and Pluronic® F68). The concentration and self-aggregation-dependent effects of surface active ophthalmic excipients on ophthalmic drug permeability were studied from the concentration region where only dissolved monomer molecules of surface active ophthalmic excipients exist, as well as the concentration region in which aggregates of variable size and dispersion are spontaneously formed. Neither the surface active ophthalmic excipients nor the ophthalmic drugs at all concentrations that were tested significantly affected the barrier properties of both corneal models, as assessed by transepithelial electrical resistance (TEER) monitoring during the permeability experiments. The lowest concentration of all investigated surface active ophthalmic excipients did not significantly affect the ophthalmic drug permeability across both of the corneal models that were used. For three ophthalmic drugs (i.e., chloramphenicol, diclofenac sodium and dexamethasone), depressed in vitro and ex vivo permeability were observed in the concentration range of either Polysorbate 80, Tyloxapol, Cremophor® EL or Pluronic® F68, at which self-aggregation is detected. The effect was the most pronounced for Cremophor® EL (1 and 2%, w/V) and was the least pronounced for Pluronic® F68 (1%, w/V). However, all surface active ophthalmic excipients over the entire concentration range that was tested did not significantly affect the in vitro

Formulation, and physical, in vitro and ex vivo evaluation of transdermal ibuprofen hydrogels containing turpentine oil as penetration enhancer.

PubMed

Khan, N R; Khan, G M; Wahab, A; Khan, A R; Hussain, A; Nawaz, A; Akhlaq, M

2011-11-01

The aim of the present study was to investigate the transdermal permeation enhancing capability of turpentine oil for ibuprofen from hydrogels. Ibuprofen 1% w/v hydrogels were developed with carboxypolymethylene with and without turpentine oil. Turpentine oil was incorporated in increasing concentrations, i.e. 0.5, 1, 1.5, 2, 2.5 and 3% of the total gel formulation, and its permeation enhancing effect was examined. Gels were examined physically for pH, viscosity, spreadability, extrudability, smoothness and appearance. To study the in vitro and ex vivo permeation potential of formulated gels, permeation studies were performed with a Franz diffusion cell using cellulose membrane and excised rabbit abdominal skin. Ibuprofen hydrogel with 3% turpentine oil showed a maximum flux of 10.87 mg/cm2/h across artificial skin and 17.26 mg/cm2/h across rabbit abdominal skin.

Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

NASA Astrophysics Data System (ADS)

Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

2016-03-01

This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

Low-frequency sonophoresis enhances rivastigmine permeation in vitro and in vivo.

PubMed

Yu, Zhen-wei; Liang, Yi; Liang, Wen-quan

2015-06-01

We investigated the enhancement effect of low-frequency sonophoresis on transdermal permeation of rivastigmine in vitro and in vivo. The in vitro permeation study showed that sonophoresis increased steady-state transdermal flux 0.31 ± 0.03 μg x cm(-2) x h(-1) and the extent of rivastigmine permeation 6.00 ± 0.56 μg x cm(-2) though excised skin (both P < 0.01). In the in vivo experiment, the C(max) 0.83 ± 0.16 μg x mL(-1) and AUC(0 --> 24 h) 12.35 ± 1.99 μg x h x mL(-1) of the sonophoresis group was also significantly higher than that of the control group (both P < 0.01). These data suggest that low-frequency sonophoresis could be an effective method to enhance rivastigmine permeation.

Matrix-type transdermal films to enhance simvastatin ex vivo skin permeability.

PubMed

El-Say, Khalid M; Ahmed, Osama A A; Aljaeid, Bader M; Zidan, Ahmed S

2017-06-01

This study aimed at employing Plackett-Burman design in screening formulation variables that affect quality of matrix-type simvastatin (SMV) transdermal film. To achieve this goal, 12 formulations were prepared by casting method. The investigated variables were Eudragit RL percentage, polymer mixture percentage, plasticizer type, plasticizer percentage, enhancer type, enhancer percentage and dichloromethane fraction in organic phase. The films were evaluated for physicochemical properties and ex vivo SMV permeation. SMV initial, delayed flux, diffusivity and permeability coefficient were calculated on the delayed flux phase with constraint to minimize the initial flux and approaching steady-state flux. The obtained results revealed flat films with homogeneous distribution of SMV within the films. Thickness values changed from 65 to 180 μm by changing the factors' combinations. Most of the permeation profiles showed sustained release feature with fast permeation phase followed by slow phase. Analysis of variance (ANOVA) showed significant effects (p < 0.05) of the investigated variables on the responses with Prob > F values of 0.0147, 0.0814, 0.0063 and 0.0142 for the initial and delayed fluxes, permeability coefficients and diffusivities, respectively. The findings of screening study showed the importance of the significant variables to be scaled up for full optimization study as a promising alternative drug delivery system.

Behavior of Tip-Steerable Needles in ex vivo and in vivo Tissue

PubMed Central

Majewicz, Ann; Marra, Steven P.; van Vledder, Mark G.; Lin, MingDe; Choti, Michael A.; Song, Danny Y.; Okamura, Allison M.

2012-01-01

Robotic needle steering is a promising technique to improve the effectiveness of needle-based clinical procedures, such as biopsies and ablation, by computer-controlled, curved insertions of needles within solid organs. In this paper, we explore the capabilities, challenges, and clinical relevance of asymmetric-tip needle steering though experiments in ex vivo and in vivo tissue. We evaluate the repeatability of needle insertion in inhomogeneous biological tissue and compare ex vivo and in vivo needle curvature and insertion forces. Steerable needles curved more in kidney than in liver and prostate, likely due to differences in tissue properties. Pre-bent needles produced higher insertion forces in liver and more curvature in vivo than ex vivo. When compared to straight stainless steel needles, steerable needles did not cause a measurable increase in tissue damage and did not exert more force during insertion. The minimum radius of curvature achieved by pre-bent needles was 5.23 cm in ex vivo tissue, and 10.4 cm in in vivo tissue. The curvatures achieved by bevel tip needles were negligible for in vivo tissue. The minimum radius of curvature for bevel tip needles in ex vivo tissue was 16.4 cm; however, about half of the bevel tip needles had negligible curvatures. We also demonstrate a potential clinical application of needle steering by targeting and ablating overlapping regions of cadaveric canine liver. PMID:22711767

Comparison of in vivo and ex vivo viscoelastic behavior of the spinal cord.

PubMed

Ramo, Nicole L; Shetye, Snehal S; Streijger, Femke; Lee, Jae H T; Troyer, Kevin L; Kwon, Brian K; Cripton, Peter; Puttlitz, Christian M

2018-03-01

Despite efforts to simulate the in vivo environment, post-mortem degradation and lack of blood perfusion complicate the use of ex vivo derived material models in computational studies of spinal cord injury. In order to quantify the mechanical changes that manifest ex vivo, the viscoelastic behavior of in vivo and ex vivo porcine spinal cord samples were compared. Stress-relaxation data from each condition were fit to a non-linear viscoelastic model using a novel characterization technique called the direct fit method. To validate the presented material models, the parameters obtained for each condition were used to predict the respective dynamic cyclic response. Both ex vivo and in vivo samples displayed non-linear viscoelastic behavior with a significant increase in relaxation with applied strain. However, at all three strain magnitudes compared, ex vivo samples experienced a higher stress and greater relaxation than in vivo samples. Significant differences between model parameters also showed distinct relaxation behaviors, especially in non-linear relaxation modulus components associated with the short-term response (0.1-1 s). The results of this study underscore the necessity of utilizing material models developed from in vivo experimental data for studies of spinal cord injury, where the time-dependent properties are critical. The ability of each material model to accurately predict the dynamic cyclic response validates the presented methodology and supports the use of the in vivo model in future high-resolution finite element modeling efforts. Neural tissues (such as the brain and spinal cord) display time-dependent, or viscoelastic, mechanical behavior making it difficult to model how they respond to various loading conditions, including injury. Methods that aim to characterize the behavior of the spinal cord almost exclusively use ex vivo cadaveric or animal samples, despite evidence that time after death affects the behavior compared to that in a living

Enhanced transdermal delivery of ondansetron using nanovesicular systems: Fabrication, characterization, optimization and ex-vivo permeation study-Box-Cox transformation practical example.

PubMed

Habib, Basant A; Sayed, Sinar; Elsayed, Ghada M

2018-03-30

This study aimed to formulate suitable nanovesicles (NVs) for transdermal delivery of Ondansetron. It also illustrated a practical example for the importance of Box-Cox transformation. A 2 3 full factorial design was used to enable testing transfersomes, ethosomes, and transethosomes of Ondansetron simultaneously. The independent variables (IVs) studied were sodium taurocholate amount, ethanol volume in hydration medium and sonication time. The studied dependent variables (DVs) were: particle size (PS), zeta potential (ZP) and entrapment efficiency (EE). Polynomial equations were used to study the influence of IVs on each DV. Numerical multiple response optimization was applied to select an optimized formula (OF) with the goals of minimizing PS and maximizing ZP absolute value and EE. Box-Cox transformation was adopted to enable modeling PS raised to the power of 1.2 with an excellent prediction R 2 of 1.000. ZP and EE were adequately represented directly with prediction R 2 of 0.9549 and 0.9892 respectively. Response surface plots helped in explaining the influence of IVs on each DV. Two-sided 95% prediction interval test and percent deviation of actual values from predicted ones proved the validity of the elucidated models. The OF was a transfersomal formula with desirability of 0.866 and showed promising results in ex-vivo permeation study. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of the in vivo and ex vivo optical properties in a mouse ear model

NASA Astrophysics Data System (ADS)

Salomatina, E.; Yaroslavsky, A. N.

2008-06-01

Determination of in vivo optical properties is a challenging problem. Absorption and scattering measured ex vivo are often used for in vivo applications. To investigate the validity of this approach, we have obtained and compared the optical properties of mouse ears in vivo and ex vivo in the spectral range from 370 to 1650 nm. Integrating sphere spectrophotometry in combination with the inverse Monte Carlo technique was employed to determine absorption coefficients, μa, scattering coefficients, μs, and anisotropy factors, g. Two groups of mice were used for the study. The first group was measured in vivo and ex vivo within 5-10 min post mortem. The second group was measured in vivo and ex vivo every 24 h for up to 72 h after sacrifice. Between the measurements the tissues were kept at 4 °C wrapped in a gauze moistened with saline solution. Then the specimens were frozen at -25 °C for 40 min, thawed and measured again. The results indicate that the absorption coefficients determined in vivo and ex vivo within 5-10 min post mortem differed considerably only in the spectral range dominated by hemoglobin. These changes can be attributed to rapid deoxygenation of tissue and blood post mortem. Absorption coefficients determined ex vivo up to 72 h post mortem decreased gradually with time in the spectral regions dominated by hemoglobin and water, which can be explained by the continuing loss of blood. Absorption properties of the frozen-thawed ex vivo tissues showed increase in oxygenation, which is likely caused by the release of hemoglobin from hemolyzed erythrocytes. Scattering of the ex vivo tissues decreased gradually with time in the entire spectral range due to the continuing loss of blood and partial cell damage. Anisotropy factors did not change considerably.

Evaluation of the in vivo and ex vivo optical properties in a mouse ear model.

PubMed

Salomatina, E; Yaroslavsky, A N

2008-06-07

Determination of in vivo optical properties is a challenging problem. Absorption and scattering measured ex vivo are often used for in vivo applications. To investigate the validity of this approach, we have obtained and compared the optical properties of mouse ears in vivo and ex vivo in the spectral range from 370 to 1650 nm. Integrating sphere spectrophotometry in combination with the inverse Monte Carlo technique was employed to determine absorption coefficients, mu(a), scattering coefficients, mu(s), and anisotropy factors, g. Two groups of mice were used for the study. The first group was measured in vivo and ex vivo within 5-10 min post mortem. The second group was measured in vivo and ex vivo every 24 h for up to 72 h after sacrifice. Between the measurements the tissues were kept at 4 degrees C wrapped in a gauze moistened with saline solution. Then the specimens were frozen at -25 degrees C for 40 min, thawed and measured again. The results indicate that the absorption coefficients determined in vivo and ex vivo within 5-10 min post mortem differed considerably only in the spectral range dominated by hemoglobin. These changes can be attributed to rapid deoxygenation of tissue and blood post mortem. Absorption coefficients determined ex vivo up to 72 h post mortem decreased gradually with time in the spectral regions dominated by hemoglobin and water, which can be explained by the continuing loss of blood. Absorption properties of the frozen-thawed ex vivo tissues showed increase in oxygenation, which is likely caused by the release of hemoglobin from hemolyzed erythrocytes. Scattering of the ex vivo tissues decreased gradually with time in the entire spectral range due to the continuing loss of blood and partial cell damage. Anisotropy factors did not change considerably.

Lipid drug conjugate nanoparticle as a novel lipid nanocarrier for the oral delivery of decitabine: ex vivo gut permeation studies

NASA Astrophysics Data System (ADS)

Neupane, Yub Raj; Sabir, M. D.; Ahmad, Nafees; Ali, Mushir; Kohli, Kanchan

2013-10-01

The purpose of this study was to develop lipid drug conjugate (LDC) nanoparticles of decitabine (DCB) using stearic acid as a lipid to increase the permeability of the drug along with its protection from chemical degradation. The LDC was prepared by salt formation of DCB with stearic acid and followed by cold homogenization technique to produce the LDC nanoparticles. The role of key independent variables influencing on dependent variables were determined by using a Box-Behnken design. The optimized batch revealed spherical morphology under TEM analysis with particle size of 202.6 ± 1.65 nm and 0.334 ± 0.987 PDI. The zeta potential and %EE were found to be -33.6 ± 0.845 mV and 68.89% ± 0.59 respectively. Lyophilized powder showed the crystalline structure under DSC analysis. In vitro release studies showed the initial burst release followed by a sustained release up to 24 h in PBS pH 7.4 and the data were further studied using release kinetic models which revealed the first-order model as a best-fitting model. Ex vivo gut permeation studies proved that the formulation containing lipid and surfactants has a higher permeability than the plain drug solution with nearly fourfold increase in the apparent permeability coefficients. Finally, LDC nanoparticles prepared by using stearic acid as a lipid and surfactants as Tween 80, Poloxamer 188, and Labrasol in equal ratio possess high potential for the oral delivery of hydrophilic drugs.

Ex-vivo machine perfusion for kidney preservation.

PubMed

Hamar, Matyas; Selzner, Markus

2018-06-01

Machine perfusion is a novel strategy to decrease preservation injury, improve graft assessment, and increase organ acceptance for transplantation. This review summarizes the current advances in ex-vivo machine-based kidney preservation technologies over the last year. Ex-vivo perfusion technologies, such as hypothermic and normothermic machine perfusion and controlled oxygenated rewarming, have gained high interest in the field of organ preservation. Keeping kidney grafts functionally and metabolically active during the preservation period offers a unique chance for viability assessment, reconditioning, and organ repair. Normothermic ex-vivo kidney perfusion has been recently translated into clinical practice. Preclinical results suggest that prolonged warm perfusion appears superior than a brief end-ischemic reconditioning in terms of renal function and injury. An established standardized protocol for continuous warm perfusion is still not available for human grafts. Ex-vivo machine perfusion represents a superior organ preservation method over static cold storage. There is still an urgent need for the optimization of the perfusion fluid and machine technology and to identify the optimal indication in kidney transplantation. Recent research is focusing on graft assessment and therapeutic strategies.

Ex vivo skin absorption of terpenes from Vicks VapoRub ointment.

PubMed

Cal, Krzysztof; Sopala, Monika

2008-08-01

The pharmaceutical market offers a wide range of inhalant drug products applied on the skin that contain essential oils and/or their isolated compounds, i.e. terpenes. Because there are few data concerning the skin penetration of terpenes, especially from complex carriers, the goal of this study was to determine the ex vivo skin absorption kinetics of chosen terpenes, namely eucalyptol, menthol, camphor, alpha-pinene, and beta-pinene, from the product Vicks VapoRub. Human cadaver skin was placed in a flow-through diffusion chamber and the product was applied for 15, 30, and 60 min. After the application time the skin was separated into layers using a tape-stripping technique: three fractions of stratum corneum and epidermis with dermis, and terpenes amounts in the samples were determined by gas-chromatography. The investigated terpenes showed different absorption characteristics related to their physicochemical properties and did not permeate through the skin into the acceptor fluid. Eucalyptol had the largest total accumulation in the stratum corneum and in the epidermis with dermis, while alpha-pinene penetrated into the skin in the smallest amount. The short time in which saturation of the stratum corneum with the terpenes occurred and the high accumulation of most of the investigated terpenes in the skin layers proved that these compounds easily penetrate and permeate the stratum corneum and that in vivo they may easily penetrate into the blood circulation.

3D morphological analysis of the mouse cerebral vasculature: Comparison of in vivo and ex vivo methods

PubMed Central

Steinman, Joe; Koletar, Margaret M.; Stefanovic, Bojana; Sled, John G.

2017-01-01

Ex vivo 2-photon fluorescence microscopy (2PFM) with optical clearing enables vascular imaging deep into tissue. However, optical clearing may also produce spherical aberrations if the objective lens is not index-matched to the clearing material, while the perfusion, clearing, and fixation procedure may alter vascular morphology. We compared in vivo and ex vivo 2PFM in mice, focusing on apparent differences in microvascular signal and morphology. Following in vivo imaging, the mice (four total) were perfused with a fluorescent gel and their brains fructose-cleared. The brain regions imaged in vivo were imaged ex vivo. Vessels were segmented in both images using an automated tracing algorithm that accounts for the spatially varying PSF in the ex vivo images. This spatial variance is induced by spherical aberrations caused by imaging fructose-cleared tissue with a water-immersion objective. Alignment of the ex vivo image to the in vivo image through a non-linear warping algorithm enabled comparison of apparent vessel diameter, as well as differences in signal. Shrinkage varied as a function of diameter, with capillaries rendered smaller ex vivo by 13%, while penetrating vessels shrunk by 34%. The pial vasculature attenuated in vivo microvascular signal by 40% 300 μm below the tissue surface, but this effect was absent ex vivo. On the whole, ex vivo imaging was found to be valuable for studying deep cortical vasculature. PMID:29053753

Hematopoietic Stem Cells: Transcriptional Regulation, Ex Vivo Expansion and Clinical Application

PubMed Central

Aggarwal, R.; Lu, J.; Pompili, V.J.; Das, H.

2012-01-01

Maintenance of ex vivo hematopoietic stem cells (HSC) pool and its differentiated progeny is regulated by complex network of transcriptional factors, cell cycle proteins, extracellular matrix, and their microenvironment through an orchestrated fashion. Strides have been made to understand the mechanisms regulating in vivo quiescence and proliferation of HSCs to develop strategies for ex vivo expansion. Ex vivo expansion of HSCs is important to procure sufficient number of stem cells and as easily available source for HSC transplants for patients suffering from hematological disorders and malignancies. Our lab has established a nanofiber-based ex vivo expansion strategy for HSCs, while preserving their stem cell characteristics. Ex vivo expanded cells were also found biologically functional in various disease models. However, the therapeutic potential of expanded stem cells at clinical level still needs to be verified. This review outlines transcriptional factors that regulate development of HSCs and their commitment, genes that regulate cell cycle status, studies that attempt to develop an effective and efficient protocol for ex vivo expansion of HSCs and application of HSC in various non-malignant and malignant disorders. Overall the goal of the current review is to deliver an understanding of factors that are critical in resolving the challenges that limit the expansion of HSCs in vivo and ex vivo. PMID:22082480

Reproducibility and Variation of Diffusion Measures in the Squirrel Monkey Brain, In Vivo and Ex Vivo

PubMed Central

Schilling, Kurt; Gao, Yurui; Stepniewska, Iwona; Choe, Ann S; Landman, Bennett A; Anderson, Adam W

2016-01-01

Purpose Animal models are needed to better understand the relationship between diffusion MRI (dMRI) and the underlying tissue microstructure. One promising model for validation studies is the common squirrel monkey, Saimiri sciureus. This study aims to determine (1) the reproducibility of in vivo diffusion measures both within and between subjects; (2) the agreement between in vivo and ex vivo data acquired from the same specimen and (3) normal diffusion values and their variation across brain regions. Methods Data were acquired from three healthy squirrel monkeys, each imaged twice in vivo and once ex vivo. Reproducibility of fractional anisotropy (FA), mean diffusivity (MD), and principal eigenvector (PEV) was assessed, and normal values were determined both in vivo and ex vivo. Results The calculated coefficients of variation (CVs) for both intra-subject and inter-subject MD were below 10% (low variability) while FA had a wider range of CVs, 2–14% intra-subject (moderate variability), and 3–31% inter-subject (high variability). MD in ex vivo tissue was lower than in vivo (30%–50% decrease), while FA values increased in all regions (30–39% increase). The mode of angular differences between in vivo and ex vivo PEVs was 12 degrees. Conclusion This study characterizes the diffusion properties of the squirrel monkey brain and serves as the groundwork for using the squirrel monkey, both in vivo and ex vivo, as a model for diffusion MRI studies. PMID:27587226

Ex Vivo Artifacts and Histopathologic Pitfalls in the Lung.

PubMed

Thunnissen, Erik; Blaauwgeers, Hans J L G; de Cuba, Erienne M V; Yick, Ching Yong; Flieder, Douglas B

2016-03-01

Surgical and pathologic handling of lung physically affects lung tissue. This leads to artifacts that alter the morphologic appearance of pulmonary parenchyma. To describe and illustrate mechanisms of ex vivo artifacts that may lead to diagnostic pitfalls. In this study 4 mechanisms of ex vivo artifacts and corresponding diagnostic pitfalls are described and illustrated. The 4 patterns of artifacts are: (1) surgical collapse, due to the removal of air and blood from pulmonary resections; (2) ex vivo contraction of bronchial and bronchiolar smooth muscle; (3) clamping edema of open lung biopsies; and (4) spreading of tissue fragments and individual cells through a knife surface. Morphologic pitfalls include diagnostic patterns of adenocarcinoma, asthma, constrictive bronchiolitis, and lymphedema. Four patterns of pulmonary ex vivo artifacts are important to recognize in order to avoid morphologic misinterpretations.

An improved cryopreservation method for porcine buccal mucosa in ex vivo drug permeation studies using Franz diffusion cells.

PubMed

Amores, Sonia; Domenech, José; Colom, Helena; Calpena, Ana C; Clares, Beatriz; Gimeno, Álvaro; Lauroba, Jacinto

2014-08-18

The use of isolated animal models to assess percutaneous absorption of molecules is frequently reported. The porcine buccal mucosa has been proposed as a substitute for the buccal mucosa barrier on ex vivo permeability studies avoiding unnecessary sacrifice of animals. But it is not always easy to obtain fresh buccal mucosa. Consequently, human and porcine buccal mucosa is sometimes frozen and stored in liquid nitrogen, but this procedure is not always feasible. One cheaper and simpler alternative is to freeze the buccal mucosa of freshly slaughtered pigs in a mechanical freezer, using DMSO and albumin as cryoprotective agents. This study compared the ex vivo permeability parameters of propranolol hydrochloride through porcine buccal mucosa using a Franz diffusion cell system and HPLC as detection method. The freezing effects on drug permeability parameters were evaluated. Equally histological studies were performed. Furthermore, the use of the parameter transmucosal water loss (TMWL) as an indicator of the buccal mucosa integrity was evaluated just as transepidermal water loss (TEWL) is utilized for skin integrity. The results showed no difference between fresh and frozen mucosal flux, permeability coefficient or lag time of propranolol. However, statistical significant difference in TMWL between fresh and frozen mucosa was observed. Copyright © 2014 Elsevier B.V. All rights reserved.

Curcumin loaded nano globules for solubility enhancement: preparation, characterization and ex vivo release study.

PubMed

Kumar, Anil; Ahuja, Alka; Ali, Javed; Baboota, Sanjula

2012-11-01

Curcumin in spite of being an effective chemotherapeutic agent against different type of cancer, suffer from the problem of low systemic bioavailability due to low aqueous solubility, extensive intestinal metabolism and first-pass metabolism when administered via the oral route. The aim of present investigation was to evaluate the potential of nano globules based nanoemulsion formulation for the solubility enhancement of curcumin. The nano globules based formulation was developed using Labrafac Lipophile WL 1349, Unitop FFT 40, PEG 400 and distilled water as an oil, surfactant, co-surfactant and aqueous phase respectively using aqueous titration method. Furthermore, different formulations were subjected to physical stability and consequently evaluated for ex vivo permeation using small intestine. The optimized formulation had small average globule diameter of 58 nm with zeta potential of -32 mv which indicated long-term dispersion stability. The globules were spherical in shape as observed by Transmission electron microscopy. During ex vivo study, the release of curcumin from nanoemulsion was 96.21% and 98.1% in 6 h and 12 h respectively whereas CU suspension was release up to 28.2% at the end of 12 h. This indicated the enhancement of solubility of curcumin in aqueous solution which is the rate limiting step in the absorption of curcumin in the intestine.

Mechanical properties of porcine brain tissue in vivo and ex vivo estimated by MR elastography.

PubMed

Guertler, Charlotte A; Okamoto, Ruth J; Schmidt, John L; Badachhape, Andrew A; Johnson, Curtis L; Bayly, Philip V

2018-03-01

The mechanical properties of brain tissue in vivo determine the response of the brain to rapid skull acceleration. These properties are thus of great interest to the developers of mathematical models of traumatic brain injury (TBI) or neurosurgical simulations. Animal models provide valuable insight that can improve TBI modeling. In this study we compare estimates of mechanical properties of the Yucatan mini-pig brain in vivo and ex vivo using magnetic resonance elastography (MRE) at multiple frequencies. MRE allows estimations of properties in soft tissue, either in vivo or ex vivo, by imaging harmonic shear wave propagation. Most direct measurements of brain mechanical properties have been performed using samples of brain tissue ex vivo. It has been observed that direct estimates of brain mechanical properties depend on the frequency and amplitude of loading, as well as the time post-mortem and condition of the sample. Using MRE in the same animals at overlapping frequencies, we observe that porcine brain tissue in vivo appears stiffer than porcine brain tissue samples ex vivo at frequencies of 100 Hz and 125 Hz, but measurements show closer agreement at lower frequencies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Experience with the first 50 ex vivo lung perfusions in clinical transplantation.

PubMed

Cypel, Marcelo; Yeung, Jonathan C; Machuca, Tiago; Chen, Manyin; Singer, Lianne G; Yasufuku, Kazuhiro; de Perrot, Marc; Pierre, Andrew; Waddell, Thomas K; Keshavjee, Shaf

2012-11-01

Normothermic ex vivo lung perfusion is a novel method to evaluate and improve the function of injured donor lungs. We reviewed our experience with 50 consecutive transplants after ex vivo lung perfusion. A retrospective study using prospectively collected data was performed. High-risk brain death donor lungs (defined as Pao(2)/Fio(2) <300 mm Hg or lungs with radiographic or clinical findings of pulmonary edema) and lungs from cardiac death donors were subjected to 4 to 6 hours of ex vivo lung perfusion. Lungs that achieved stable airway and vascular pressures and Pao(2)/Fio(2) greater than 400 mm Hg during ex vivo lung perfusion were transplanted. The primary end point was the incidence of primary graft dysfunction grade 3 at 72 hours after transplantation. End points were compared with lung transplants not treated with ex vivo lung perfusion (controls). A total of 317 lung transplants were performed during the study period (39 months). Fifty-eight ex vivo lung perfusion procedures were performed, resulting in 50 transplants (86% use). Of these, 22 were from cardiac death donors and 28 were from brain death donors. The mean donor Pao(2)/Fio(2) was 334 mm Hg in the ex vivo lung perfusion group and 452 mm Hg in the control group (P = .0001). The incidence of primary graft dysfunction grade 3 at 72 hours was 2% in the ex vivo lung perfusion group and 8.5% in the control group (P = .14). One patient (2%) in the ex vivo lung perfusion group and 7 patients (2.7%) in the control group required extracorporeal lung support for primary graft dysfunction (P = 1.00). The median time to extubation, intensive care unit stay, and hospital length of stay were 2, 4, and 20 days, respectively, in the ex vivo lung perfusion group and 2, 4, and 23 days, respectively, in the control group (P > .05). Thirty-day mortality (4% in the ex vivo lung perfusion group and 3.5% in the control group, P = 1.00) and 1-year survival (87% in the ex vivo lung perfusion group and 86% in the control

Optimized nano-transfersomal films for enhanced sildenafil citrate transdermal delivery: ex vivo and in vivo evaluation

PubMed Central

Badr-Eldin, Shaimaa M; Ahmed, Osamaa AA

2016-01-01

Sildenafil citrate (SLD) is a selective cyclic guanosine monophosphate-specific phosphodiesterase type 5 inhibitor used for the oral treatment of erectile dysfunction and, more recently, for other indications, including pulmonary hypertension. The challenges facing the oral administration of the drug include poor bioavailability and short duration of action that requires frequent administration. Thus, the objective of this work is to formulate optimized SLD nano-transfersomal transdermal films with enhanced and controlled permeation aiming at surmounting the previously mentioned challenges and hence improving the drug bioavailability. SLD nano-transfersomes were prepared using modified lipid hydration technique. Central composite design was applied for the optimization of SLD nano-transfersomes with minimized vesicular size. The independent variables studied were drug-to-phospholipid molar ratio, surfactant hydrophilic lipophilic balance, and hydration medium pH. The optimized SLD nano-transfersomes were developed and evaluated for vesicular size and morphology and then incorporated into hydroxypropyl methyl cellulose transdermal films. The optimized transfersomes were unilamellar and spherical in shape with vesicular size of 130 nm. The optimized SLD nano-transfersomal films exhibited enhanced ex vivo permeation parameters with controlled profile compared to SLD control films. Furthermore, enhanced bioavailability and extended absorption were demonstrated by SLD nano-transfersomal films as reflected by their significantly higher maximum plasma concentration (Cmax) and area under the curve and longer time to maxi mum plasma concentration (Tmax) compared to control films. These results highlighted the potentiality of optimized SLD nano-transfersomal films to enhance the transdermal permeation and the bioavailability of the drug with the possible consequence of reducing the dose and administration frequency. PMID:27103786

Optimized nano-transfersomal films for enhanced sildenafil citrate transdermal delivery: ex vivo and in vivo evaluation.

PubMed

Badr-Eldin, Shaimaa M; Ahmed, Osamaa Aa

2016-01-01

Sildenafil citrate (SLD) is a selective cyclic guanosine monophosphate-specific phosphodiesterase type 5 inhibitor used for the oral treatment of erectile dysfunction and, more recently, for other indications, including pulmonary hypertension. The challenges facing the oral administration of the drug include poor bioavailability and short duration of action that requires frequent administration. Thus, the objective of this work is to formulate optimized SLD nano-transfersomal transdermal films with enhanced and controlled permeation aiming at surmounting the previously mentioned challenges and hence improving the drug bioavailability. SLD nano-transfersomes were prepared using modified lipid hydration technique. Central composite design was applied for the optimization of SLD nano-transfersomes with minimized vesicular size. The independent variables studied were drug-to-phospholipid molar ratio, surfactant hydrophilic lipophilic balance, and hydration medium pH. The optimized SLD nano-transfersomes were developed and evaluated for vesicular size and morphology and then incorporated into hydroxypropyl methyl cellulose transdermal films. The optimized transfersomes were unilamellar and spherical in shape with vesicular size of 130 nm. The optimized SLD nano-transfersomal films exhibited enhanced ex vivo permeation parameters with controlled profile compared to SLD control films. Furthermore, enhanced bioavailability and extended absorption were demonstrated by SLD nano-transfersomal films as reflected by their significantly higher maximum plasma concentration (C max) and area under the curve and longer time to maxi mum plasma concentration (T max) compared to control films. These results highlighted the potentiality of optimized SLD nano-transfersomal films to enhance the transdermal permeation and the bioavailability of the drug with the possible consequence of reducing the dose and administration frequency.

Ex Vivo Lung Perfusion: Establishment and Operationalization in Iran.

PubMed

Shafaghi, Shadi; Abbasi Dezfuli, Azizollah; Ansari Aval, Zahra; Sheikhy, Kambiz; Farzanegan, Behrooz; Mortaz, Esmaeil; Emami, Habib; Aigner, Clemens; Hosseini-Baharanchi, Fatemeh Sadat; Najafizadeh, Katayoun

2017-02-01

Although the number of lung transplants is limited because of general shortage of organ donors, ex vivo lung perfusion is a novel method with 2 main benefits, including better evaluation of lung potential and recovery of injured lungs. The main aim of this study was to establish and operationalize ex vivo lung perfusion as the first experience in Iran. This was a prospective operational research study on 5 cases, including 1 pig from Vienna Medical University and 4 patients from Masih Daneshvari Hospital. All organ donations from brain dead donors were evaluated according to lung transplant or ex vivo lung perfusion criteria from May 2013 to July 2015 in Tehran, Iran. If a donor did not have any sign of severe chest trauma or pneumonia but had poor oxygenation due to possible atelectasis or neurogenic pulmonary edema, their lungs were included for ex vivo lung perfusion. A successful trend in the difference between the pulmonary arterial Po2 and the left atrial Po2 was observed, as well as an increasing pattern in other functional parameters, including dynamic lung compliance and a decreasing trend in pulmonary vascular resistance. These initial trials indicate that ex vivo lung perfusion can lead to remarkable progress in lung transplant in Iran. They also provide several important pieces of guidance for successful ex vivo lung perfusion, including the necessity of following standard lung retrieval procedures and monitoring temperature and pressure precisely. The development of novel methods can provide opportunities for further research studies on lungs of deceased donors and lead to undiscovered findings. By keeping this science up to date in Iran and developing such new and creative methods, we can reveal effective strategies to promote the quality of donor lungs to support patients on transplant wait lists.

Antimicrobial Blue Light Therapy for Infectious Keratitis: Ex Vivo and In Vivo Studies.

PubMed

Zhu, Hong; Kochevar, Irene E; Behlau, Irmgard; Zhao, Jie; Wang, Fenghua; Wang, Yucheng; Sun, Xiaodong; Hamblin, Michael R; Dai, Tianhong

2017-01-01

To investigate the effectiveness of antimicrobial blue light (aBL) as an alternative or adjunctive therapeutic for infectious keratitis. We developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis. A bioluminescent strain of Pseudomonas aeruginosa was used as the causative pathogen, allowing noninvasive monitoring of the extent of infection in real time via bioluminescence imaging. Quantitation of bacterial luminescence was correlated to colony-forming units (CFU). Using the ex vivo and in vivo models, the effectiveness of aBL (415 nm) for the treatment of keratitis was evaluated as a function of radiant exposure when aBL was delivered at 6 or 24 hours after bacterial inoculation. The aBL exposures calculated to reach the retina were compared to the American National Standards Institute standards to estimate aBL retinal safety. Pseudomonas aeruginosa keratitis fully developed in both the ex vivo and in vivo models at 24 hours post inoculation. Bacterial luminescence in the infected corneas correlated linearly to CFU (R2 = 0.921). Bacterial burden in the infected corneas was rapidly and significantly reduced (>2-log10) both ex vivo and in vivo after a single exposure of aBL. Recurrence of infection was observed in the aBL-treated mice at 24 hours after aBL exposure. The aBL toxicity to the retina is largely dependent on the aBL transmission of the cornea. Antimicrobial blue light is a potential alternative or adjunctive therapeutic for infectious keratitis. Further studies of corneal and retinal safety using large animal models, in which the ocular anatomies are similar to that of humans, are warranted.

Design and elaboration of freeze-dried PLGA nanoparticles for the transcorneal permeation of carprofen: Ocular anti-inflammatory applications.

PubMed

Parra, Alexander; Mallandrich, Mireia; Clares, Beatriz; Egea, María A; Espina, Marta; García, María L; Calpena, Ana C

2015-12-01

This work aimed the design and development of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) for the ocular delivery of Carprofen (CP) by a central rotatable composite design 2(3)+ star. NPs showed adequate size for ocular administration (189.50 ± 1.67 nm), low polydispersity (0.01 ± 0.01), negative charge surface (-22.80 ± 0.66 mV) and optimal entrapment efficiency (74.70 ± 0.95%). Physicochemical analysis confirmed that CP was dispersed inside the NPs. The drug release followed a first order kinetic model providing greater sustained CP release after lyophilization. Ex vivo permeation analysis through isolated rabbit cornea revealed that a sufficient amount of CP was retained in the tissue avoiding excessive permeation and thus, potential systemic levels. Ex vivo ocular tolerance results showed no signs of ocular irritancy, which was also confirmed by in vivo Draize test. In vivo ocular anti-inflammatory efficacy test confirmed an optimal efficacy of NPs and its potential application in eye surgery. Copyright © 2015 Elsevier B.V. All rights reserved.

[Net power and energy of cooled antenna microwave ablation:ex vivo versus in vivo results in porcine liver].

PubMed

Jiang, Hua; Fan, Wei-jun; Zhang, Liang; Li, Xin; Zhang, Jian-lei

2012-09-18

To explore the net power and net energy of a cooled antenna radiator in ex vivo and in vivo porcine livers. All animal experiments complied with the guidelines of our animal use committee. Microwave ablation (MWA) was performed in ex vivo and in vivo porcine livers with a cooled-shaft antenna in different microwave ablation parameter groups (50, 80 and 110 W for 10 min). The energy losses from the microwave antenna or cables were calculated. And the net power, net energy and the relationship between net power and power readout were determined. When the power displayed by the machine indicated 50 W, 80 W and 110 W, the net power during MWA was 31.3 ± 0.6, 47.3 ± 0.8 and 62.1 ± 0.9 W ex vivo and 31.8 ± 0.8, 47.4 ± 0.3 and 61.7 ± 1.5 W in vivo. For the same power readout, the ex vivo or in vivo effective power was the same (P = 0.841, P = 0.133, P = 0.551). For both ex vivo and in vivo experiments, the ratio of microwave antenna energy loss to microwave antenna input energy was relatively constant (P = 0.613, 0.326). For the same treatment time and net power, the difference was significant between ex vivo and in vivo ablation volumes (P = 0.001, 0.006, 0.001). Using net power as a reference during MWA is more accurate compared to the traditional power readout. And net energy offers a more realistic reflection of MWA energy in tissues.

[Anti-platelet actions of salicylates: in vivo, ex vivo and in vitro effects of choline salicylate].

PubMed

Irino, O; Saitoh, K; Ohkubo, K

1985-07-01

Effects of choline salicylate, sodium salicylate, choline chloride and acetylsalicylic acid on platelet aggregation in vivo, ex vivo and in vitro in mice were studied. These drugs all inhibited adenosine diphosphate (ADP)-induced respiratory depression, which is closely related to platelet aggregation in vivo, with choline salicylate showing the strongest inhibitory effect. Choline salicylate had a tendency to reduce the mortality of animals injected intravenously with endotoxin, but the other drugs had no such effect. The inhibitory effects of these drugs on ADP-induced platelet aggregation ex vivo were in the order of choline salicylate greater than acetylsalicylic acid congruent to sodium salicylate greater than choline chloride congruent to no effect, and plasma concentrations of protein-unbound salicylic acid at 1 hr after oral administration of drugs were in the order of choline salicylate greater than acetylsalicylic acid congruent to sodium salicylate. The in vitro effects of these drugs were in the order of choline salicylate congruent to sodium salicylate greater than choline chloride congruent to acetylsalicylic acid congruent to no effect. Therefore, it was considered that salicylic acid played an important role on the in vivo, ex vivo and in vitro effects of choline salicylate and that choline increased plasma concentrations of salicylic acid and consequently enhanced the in vivo and ex vivo effects of salicylic acid. Furthermore, the ex vivo effects of choline salicylate were found when ADP-induced platelet aggregation was measured with platelet-rich plasma prepared from blood collected with heparin as anti-coagulant, but not when blood was collected with citrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Lyophilized insulin nanoparticles prepared from quaternized N-aryl derivatives of chitosan as a new strategy for oral delivery of insulin: in vitro, ex vivo and in vivo characterizations.

PubMed

Mahjub, Reza; Radmehr, Moojan; Dorkoosh, Farid Abedin; Ostad, Seyed Naser; Rafiee-Tehrani, Morteza

2014-12-01

from quaternized aromatic derivatives of chitosan. In vivo data showed significantly higher insulin intestinal absorption in nanoparticles prepared from methylated N-(4-N, N-dimethylaminobenzyl) chitosan nanoparticles compared to trimethyl chitosan. These data obtained demonstrated that as the result of optimized physico-chemical properties, drug release rate, cytotoxicity profile, ex vivo permeation enhancement and increased in vivo absorption, nanoparticles prepared from N-aryl derivatives of chitosan can be considered as valuable method for the oral delivery of insulin.

Radical protection in the visible and infrared by a hyperforin-rich cream--in vivo versus ex vivo methods.

PubMed

Arndt, Sophia; Haag, Stefan F; Kleemann, Anke; Lademann, Juergen; Meinke, Martina C

2013-05-01

The formation of radicals plays an important role in the development of atopic eczema or barrier-disrupted skin. We evaluated the radical scavenging effect of a cream containing a Hypericum perforatum extract rich in hyperforin in a double-blind placebo-controlled study on 11 healthy volunteers. Electron paramagnetic resonance spectroscopy was applied to determine radical formation during VIS/NIR irradiation of the inner forearm. The results were compared to ex vivo investigations on excised porcine ear skin after a single application of the creams. The non-treated skin was measured as control. The absolute values and the kinetics are not comparable for ex vivo and in vivo radical formation. Whereas in vivo, the radical production decreases with time, it remains stable ex vivo over the investigated timescale. Nevertheless, ex vivo methods could be developed to estimate the protection efficiency of creams. In vivo as well as ex vivo, the radical formation could be reduced by almost 80% when applying the hyperforin-rich cream onto the skin, whereas placebo resulted in about 60%. In vivo, a daylong protection effect could be validated after a 4-week application time of the cream indicating that a regular application is necessary to obtain the full effect. © 2013 John Wiley & Sons A/S.

In vivo and ex vivo imaging with ultrahigh resolution full-field OCT

NASA Astrophysics Data System (ADS)

Grieve, Kate; Moneron, Gael; Schwartz, Wilfrid; Boccara, Albert C.; Dubois, Arnaud

2005-08-01

Imaging of in vivo and ex vivo biological samples using full-field optical coherence tomography is demonstrated. Three variations on the original full-field optical coherence tomography instrument are presented, and evaluated in terms of performance. The instruments are based on the Linnik interferometer illuminated by a white light source. Images in the en face orientation are obtained in real-time without scanning by using a two-dimensional parallel detector array. An isotropic resolution capability better than 1 μm is achieved thanks to the use of a broad spectrum source and high numerical aperture microscope objectives. Detection sensitivity up to 90 dB is demonstrated. Image acquisition times as short as 10 μs per en face image are possible. A variety of in vivo and ex vivo imaging applications is explored, particularly in the fields of embryology, ophthalmology and botany.

Fluorophore-labeling of core-crosslinked polymeric micelles for multimodal in vivo and ex vivo optical imaging

PubMed Central

Shi, Yang; Kunjachan, Sijumon; Wu, Zhuojun; Gremse, Felix; Moeckel, Diana; van Zandvoort, Marc; Kiessling, Fabian; Storm, Gert; van Nostrum, Cornelus F.; Hennink, Wim E.; Lammers, Twan

2015-01-01

Aim To enable multimodal in vivo and ex vivo optical imaging of the biodistribution and tumor accumulation of core-crosslinked polymeric micelles (CCPM). Materials & Methods mPEG-b-p(HPMAm-Lac)-based polymeric micelles, core-crosslinked via cystamine and covalently labeled with two fluorophores (Dy-676/488) were synthesized. The CCPM were intravenously injected in CT26 tumor-bearing mice. Results Upon intravenous injection, the CCPM accumulated in CT26 tumors reasonably efficiently, with values reaching ~4 %ID at 24 hours. Ex vivo TPLSM confirmed efficient extravasation of the iCCPM out of tumor blood vessels and deep penetration into the tumor interstitium. Conclusions CCPM were labeled with multiple fluorophores, and they exemplify that combining different in vivo and ex vivo optical imaging techniques is highly useful for analyzing the biodistribution and tumor accumulation of nanomedicines. PMID:25929568

Ex vivo permeation of tamoxifen and its 4-OH metabolite through rat intestine from lecithin/chitosan nanoparticles.

PubMed

Barbieri, S; Buttini, F; Rossi, A; Bettini, R; Colombo, P; Ponchel, G; Sonvico, F; Colombo, G

2015-08-01

Tamoxifen citrate is an anticancer drug slightly soluble in water. Administered orally, it shows great intra- and inter-patient variations in bioavailability. We developed a nanoformulation based on phospholipid and chitosan able to efficiently load tamoxifen and showing an enzyme triggered release. In this work the permeation of tamoxifen released from lecithin/chitosan nanoparticles across excised rat intestinal wall mounted in an Ussing chamber was investigated. Compared to tamoxifen citrate suspension, the amount of the drug permeated using the nanoformulation was increased from 1.5 to 90 times, in absence or in presence of pancreatin or lipase, respectively. It was also evidenced the formation of an active metabolite of tamoxifen, 4-hydroxy tamoxifen, however, the amount of metabolite permeated remained roughly constant in all experiments. The effect of enzymes on intestinal permeation of tamoxifen was shown only when tamoxifen-loaded nanoparticles were in intimate contact with the mucosal surface. The encapsulation of tamoxifen in lecithin/chitosan nanoparticles improved the non-metabolized drug passing through the rat intestinal tissue via paracellular transport. Copyright © 2015 Elsevier B.V. All rights reserved.

Formulation of unidirectional release buccal patches of carbamazepine and study of permeation through porcine buccal mucosa

PubMed Central

Govindasamy, Parthasarathy; Kesavan, Bhaskar Reddy; Narasimha, Jayaveera Korlakunta

2013-01-01

Objective To achieve transbuccal release of carbamazepine by loading in unidirectional release mucoadhesive buccal patches. Methods Buccal patches of carbamazepine with unidirectional drug release were prepared using hydroxypropyl methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone and ethyl cellulose by solvent casting method. Water impermeable backing layer (Pidilite® Biaxially-oriented polypropylene film) of patches provided unidirectional drug release. They were evaluated for thickness, mass uniformity, surface pH and folding endurance. Six formulations FA2, FA8, FA10, FB1, FB14 and FB16 (folding endurance above 250) were evaluated further for swelling studies, ex vivo mucoadhesive strength, ex vivo mucoadhesion time, in vitro drug release, ex vivo permeation, accelerated stability studies and FTIR and XRD spectral studies. Results The ex vivo mucoadhesion time of patches ranged between 109 min (FA10) to 126 min (FB14). The ex vivo mucoadhesive force was in the range of 0.278 to 0.479 kg/m/s. The in vitro drug release studies revealed that formulation FA8 released 84% and FB16 released 99.01% of drug in 140 min. Conclusions The prepared unidirectional buccal patches of carbamazepine provided a maximum drug release within specified mucoadhesion period and it indicates a potential alternative drug delivery system for systemic delivery of carbamazepine. PMID:24093793

In vivo and ex vivo sentinel node mapping does not identify the same lymph nodes in colon cancer.

PubMed

Andersen, Helene Schou; Bennedsen, Astrid Louise Bjørn; Burgdorf, Stefan Kobbelgaard; Eriksen, Jens Ravn; Eiholm, Susanne; Toxværd, Anders; Riis, Lene Buhl; Rosenberg, Jacob; Gögenur, Ismail

2017-07-01

Identification of lymph nodes and pathological analysis is crucial for the correct staging of colon cancer. Lymph nodes that drain directly from the tumor area are called "sentinel nodes" and are believed to be the first place for metastasis. The purpose of this study was to perform sentinel node mapping in vivo with indocyanine green and ex vivo with methylene blue in order to evaluate if the sentinel lymph nodes can be identified by both techniques. Patients with colon cancer UICC stage I-III were included from two institutions in Denmark from February 2015 to January 2016. In vivo sentinel node mapping with indocyanine green during laparoscopy and ex vivo sentinel node mapping with methylene blue were performed in all patients. Twenty-nine patients were included. The in vivo sentinel node mapping was successful in 19 cases, and ex vivo sentinel node mapping was successful in 13 cases. In seven cases, no sentinel nodes were identified. A total of 51 sentinel nodes were identified, only one of these where identified by both techniques (2.0%). In vivo sentinel node mapping identified 32 sentinel nodes, while 20 sentinel nodes were identified by ex vivo sentinel node mapping. Lymph node metastases were found in 10 patients, and only two had metastases in a sentinel node. Placing a deposit in relation to the tumor by indocyanine green in vivo or of methylene blue ex vivo could only identify sentinel lymph nodes in a small group of patients.

Expression analysis of MDR1, BCRP and MRP3 transporter proteins in different in vitro and ex vivo cornea models for drug absorption studies.

PubMed

Verstraelen, Jessica; Reichl, Stephan

2013-01-30

Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treatment. Such preclinical investigations on transcorneal drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell culture models. The data currently available on the expression of ABC transporter proteins in corneal tissue is limited or contradictory. This study describes, for the first time, the comparison of the expression of ABC transporters, in particular, MDR1, BCRP and MRP3, between human cornea cell culture models and the most commonly used ex vivo models, namely, rabbit and porcine corneas, conducted in the same laboratory. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and bidirectional permeation studies using specific substrates and inhibitors. The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the expression and activity of MDR1 transporter was confirmed, whereas human cell culture models and porcine corneas did not show MDR1 expression. However, human cornea models possessed MRP3 and BCRP expression, whereas no functional expression was found in rabbit and porcine corneas. Therefore, the translation of transcorneal permeation data from animal experiments to humans should be performed with caution. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of Curcumin loaded chitosan polymer based nanoemulsion gel: In vitro, ex vivo evaluation and in vivo wound healing studies.

PubMed

Thomas, Lydia; Zakir, Foziyah; Mirza, Mohd Aamir; Anwer, Md Khalid; Ahmad, Farhan Jalees; Iqbal, Zeenat

2017-08-01

In the present study, various nanoemulsions were prepared using Labrafac PG+Triacetin as oil, Tween 80 as a surfactant and polyethylene glycol (PEG 400) as a co-surfactant. The developed nanoemulsions (NE1-NE5) were evaluated for physicochemical characterizations and ex-vivo for skin permeation and deposition studies. The highest skin deposition was observed for NE2 with 46.07% deposition amongst all developed nanoemulsions (NE1-NE5). Optimized nanoemulsion (NE2) had vesicle size of 84.032±0.023nm, viscosity 78.23±22.2 cps, refractive index 1.404. Nanoemulsion gel were developed by incorporation of optimized nanoemulsion (NE2) into 1-3% chitosan and characterized by physical evaluation and rheological studies. Chitosan gel (2%) was found to be suitable for gelation of nanoemulsion based on its consistency, feel and ease of spreadability. The flux of nanoemulsion gel was found 68.88μg/cm 2 /h as compared to NE2 (76.05μg/cm 2 /h) is significantly lower suggesting limited skin permeation of curcumin form gel. However, the retained amount of curcumin on skin by gel formulation (980.75±88μg) is significantly higher than NE2 (771.25±67μg). Enhanced skin permeation of NE2 (46.07%) was observed when compared to nanoemulsion gel (31.25%) and plain gel (11.47%). The outcome of this study evidently points out the potential of curcumin entrapped nanoemulsion gel in wound healing. Copyright © 2017 Elsevier B.V. All rights reserved.

Susceptibility weighted imaging of cartilage canals in porcine epiphyseal growth cartilage ex vivo and in vivo.

PubMed

Nissi, Mikko J; Toth, Ferenc; Zhang, Jinjin; Schmitter, Sebastian; Benson, Michael; Carlson, Cathy S; Ellermann, Jutta M

2014-06-01

High-resolution visualization of cartilage canals has been restricted to histological methods and contrast-enhanced imaging. In this study, the feasibility of non-contrast-enhanced susceptibility weighted imaging (SWI) for visualization of the cartilage canals was investigated ex vivo at 9.4 T, further explored at 7 and 3 T and demonstrated in vivo at 7 T, using a porcine animal model. SWI scans of specimens of distal femur and humerus from 1 to 8 week-old piglets were conducted at 9.4 T using 3D-GRE sequence and SWI post-processing. The stifle joints of a 2-week old piglet were scanned ex vivo at 7 and 3 T. Finally, the same sites of a 3-week-old piglet were scanned, in vivo, at 7 T under general anesthesia using the vendor-provided sequences. High-contrast visualization of the cartilage canals was obtained ex vivo, especially at higher field strengths; the results were confirmed histologically. In vivo feasibility was demonstrated at 7 T and comparison of ex vivo scans at 3 and 7 T indicated feasibility of using SWI at 3 T. High-resolution 3D visualization of cartilage canals was demonstrated using SWI. This demonstration of fully noninvasive visualization opens new avenues to explore skeletal maturation and the role of vascular supply for diseases such as osteochondrosis. Copyright © 2013 Wiley Periodicals, Inc.

Impact of Humidity on In Vitro Human Skin Permeation Experiments for Predicting In Vivo Permeability.

PubMed

Ishida, Masahiro; Takeuchi, Hiroyuki; Endo, Hiromi; Yamaguchi, Jun-Ichi

2015-12-01

In vitro skin permeation studies have been commonly conducted to predict in vivo permeability for the development of transdermal therapeutic systems (TTSs). We clarified the impact of humidity on in vitro human skin permeation of two TTSs having different breathability and then elucidated the predictability of in vivo permeability based on in vitro experimental data. Nicotinell(®) TTS(®) 20 and Frandol(®) tape 40mg were used as model TTSs in this study. The in vitro human skin permeation experiments were conducted under humidity levels similar to those used in clinical trials (approximately 50%) as well as under higher humidity levels (approximately 95%). The skin permeability values of drugs at 95% humidity were higher than those at 50% humidity. The time profiles of the human plasma concentrations after TTS application fitted well with the clinical data when predicted based on the in vitro permeation parameters at 50% humidity. On the other hand, those profiles predicted based on the parameters at 95% humidity were overestimated. The impact of humidity was higher for the more breathable TTS; Frandol(®) tape 40mg. These results show that in vitro human skin permeation experiments should be investigated under realistic clinical humidity levels especially for breathable TTSs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

[Tartrate-sensitive and tartrate-resistant acid phosphatases in Amoeba proteus].

PubMed

Sopina, V A; Beliaeva, T N

2000-01-01

In free-living Amoeba proteus (strain B), acid phosphatase (AcP) was examined by disc-electrophoresis in polyacrylamide gel. The tartrate-sensitive amebian AcP was greatly inhibited by dithiothreitol and Cu2+, and only partly inhibited by sodium orthovanadate, ammonium molybdate, EDTA, disodium salt and Mg2+, Ca2+, Zn2+ and Mn2+. On the contrary, it appeared to be resistant to sulfhydryl reagents--4(hydroxymercury) benzoic acid, sodium salt and N-ethylmaleimide. Unlike the tartrate-sensitive enzyme, the tartrate-resistant AcP was greatly inhibited by EDTA and partly inhibited by dithiothreitol, Mg2+ and Cu2+ (Mn2+ > Cu2+), being activated by orthovanadate, molybdate, sulfhydryl reagents, Mg2+, Ca2+ and Zn2+. Both tartrate-sensitive and tartrate-resistant AcPs lack apparently free SH-groups necessary for their catalytic activities. Using 2-naphthyl phosphate as a substrate at pH 4.5, six AcP electromorphs were revealed in cytosol and sediment, four of these being most frequently localized in the former, and two in the latter. Two other AcP electromorphs were confined to the sediment only. Depending on the quantity of sedimented amoebae making a homogenate (0.5 or 2.0 cm3), that was added to Percoll solution, the lysosomal AcP fraction in polyacrylamide gel was represented by one or two tartrate-sensitive electromorphs. Therefore, tartrate-resistant AcP in A. proteus may be a lysosomal enzyme, while tartrate-resistant AcP may correspond to serine/threonine protein phosphatase.

Comparison of In Vivo and Ex Vivo MRI for the Detection of Structural Abnormalities in a Mouse Model of Tauopathy.

PubMed

Holmes, Holly E; Powell, Nick M; Ma, Da; Ismail, Ozama; Harrison, Ian F; Wells, Jack A; Colgan, Niall; O'Callaghan, James M; Johnson, Ross A; Murray, Tracey K; Ahmed, Zeshan; Heggenes, Morten; Fisher, Alice; Cardoso, M Jorge; Modat, Marc; O'Neill, Michael J; Collins, Emily C; Fisher, Elizabeth M C; Ourselin, Sébastien; Lythgoe, Mark F

2017-01-01

With increasingly large numbers of mouse models of human disease dedicated to MRI studies, compromises between in vivo and ex vivo MRI must be fully understood in order to inform the choice of imaging methodology. We investigate the application of high resolution in vivo and ex vivo MRI, in combination with tensor-based morphometry (TBM), to uncover morphological differences in the rTg4510 mouse model of tauopathy. The rTg4510 mouse also offers a novel paradigm by which the overexpression of mutant tau can be regulated by the administration of doxycycline, providing us with a platform on which to investigate more subtle alterations in morphology with morphometry. Both in vivo and ex vivo MRI allowed the detection of widespread bilateral patterns of atrophy in the rTg4510 mouse brain relative to wild-type controls. Regions of volume loss aligned with neuronal loss and pathological tau accumulation demonstrated by immunohistochemistry. When we sought to investigate more subtle structural alterations in the rTg4510 mice relative to a subset of doxycycline-treated rTg4510 mice, ex vivo imaging enabled the detection of more regions of morphological brain changes. The disadvantages of ex vivo MRI may however mitigate this increase in sensitivity: we observed a 10% global shrinkage in brain volume of the post-mortem tissues due to formalin fixation, which was most notable in the cerebellum and olfactory bulbs. However, many central brain regions were not adversely affected by the fixation protocol, perhaps due to our "in-skull" preparation. The disparity between our TBM findings from in vivo and ex vivo MRI underlines the importance of appropriate study design, given the trade-off between these two imaging approaches. We support the utility of in vivo MRI for morphological phenotyping of mouse models of disease; however, for subtler phenotypes, ex vivo offers enhanced sensitivity to discrete morphological changes.

In vivo and ex vivo characterization of a novel Er fiber laser system for fractional treatment of soft oral tissues

NASA Astrophysics Data System (ADS)

Shatilova, Ksenia; Aloian, Georgii; Karabut, Maria; Ryabova, Valentina; Yaroslavsky, Ilya V.; Altshuler, Gregory

2018-02-01

In this work, we present the first histological in vivo and ex vivo study of effects of fractional Er fiber laser (wavelength 1550 nm, peak power 25 W) on keratinized gum and alveolar mucosa for gum regeneration. Biopsy with subsequent NBTC staining was used as primary evaluation technique. Ex vivo, porcine tissue model was used. Effects of pulse energy, beam diameter, and beam divergence were investigated in detail. It has been demonstrated that under optimal conditions columns up to 800 μm in depth could be reliably produced with 130 mJ pulses. Clinically, 2 subjects were treated and 4 punch biopsies were collected. The results were compared with ex vivo data. Both ex vivo and in vivo datasets suggest feasibility of a dental fractional system intended for gum regeneration.

Gel in core carbosomes as novel ophthalmic vehicles with enhanced corneal permeation and residence.

PubMed

Moustafa, Mona A; El-Refaie, Wessam M; Elnaggar, Yosra S R; Abdallah, Ossama Y

2018-05-17

Carbopol is a good bio-adhesive polymer that increases the residence time in the eye. However, the effect of blinking and lacrimation still reduce the amount of polymer and the incorporated drug available for bioadhesion. Gel-core liposomes are advanced systems offering benefits making it a good tool for improved ocular drug delivery and residence time. Incorporation of carbopol in gel-core liposomes and their potential in ocular delivery have not so far been investigated. Fluconazole (FLZ) was selected as a challenging important ocular antifungal suffering from poor corneal permeation and short residence time. In this study, gel-core carbosomes have been elaborated as novel carbopol-based ophthalmic vehicles to solve ocular delivery obstacles of FLZ and to sustain its effect. Full in vitro appraisal was performed considering gel-core structure, entrapment efficiency, particle size and stability of the vesicles as quality attributes. Structure elucidation of the nanocarrier was performed using optical, polarizing and transmission electron microscopy before and after Triton-X100 addition. Ex-vivo ocular permeation and in vivo performance were investigated on male albino rabbits. Optimized formulation (CBS5) showed gel-core structure, nanosize (339.00 ± 5.50 nm) and not defined before (62.00% ± 1.73) entrapment efficiency. Cumulative amount of CBS5 permeated ex-vivo after 6 h, was 2.43 and 3.43 folds higher than that of conventional liposomes and FLZ suspension, respectively. In-vivo corneal permeation of CBS5 showed significantly higher AUC0-24 h (487.12 ± 74.80) compared to that of FLZ suspension (204.34 ± 7.46) with longer residence time in the eye lasts for more than 18 h. In conclusion, novel gel-core carbosomes could successfully be used as a promising delivery system for chronic ocular diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

PASSIVE CAVITATION DETECTION DURING PULSED HIFU EXPOSURES OF EX VIVO TISSUES AND IN VIVO MOUSE PANCREATIC TUMORS

PubMed Central

Li, Tong; Chen, Hong; Khokhlova, Tatiana; Wang, Yak-Nam; Kreider, Wayne; He, Xuemei; Hwang, Joo Ha

2014-01-01

Pulsed high-intensity focused ultrasound (pHIFU) has been demonstrated to enhance vascular permeability, disrupt tumor barriers and enhance drug penetration into tumor tissue through acoustic cavitation. Monitoring of cavitation activity during pHIFU treatments and knowing the ultrasound pressure levels sufficient to reliably induce cavitation in a given tissue are therefore very important. Here, three metrics of cavitation activity induced by pHIFU and evaluated by confocal passive cavitation detection were introduced: cavitation probability, cavitation persistence and the level of the broadband acoustic emissions. These metrics were used to characterize cavitation activity in several ex vivo tissue types (bovine tongue and liver and porcine adipose tissue and kidney) and gel phantoms (polyacrylamide and agarose) at varying peak-rarefactional focal pressures (1–12 MPa) during the following pHIFU protocol: frequency 1.1 MHz, pulse duration 1 ms, pulse repetition frequency 1 Hz. To evaluate the relevance of the measurements in ex vivo tissue, cavitation metrics were also investigated and compared in the ex vivo and in vivo murine pancreatic tumors that develop spontaneously in transgenic KPC mice and closely recapitulate human disease in their morphology. The cavitation threshold, defined at 50 % cavitation probability, was found to vary broadly among the investigated tissues (within 2.5–10 MPa), depending mostly on the water-lipid ratio that characterizes the tissue composition. Cavitation persistence and the intensity of broadband emissions depended both on tissue structure and lipid concentration. Both the cavitation threshold and broadband noise emission level were similar between ex vivo and in vivo pancreatic tumor tissue. The largest difference between in vivo and ex vivo settings was found in the pattern of cavitation occurrence throughout pHIFU exposure: it was sporadic in vivo, but ex vivo it decreased rapidly and stopped over the first few pulses

Passive cavitation detection during pulsed HIFU exposures of ex vivo tissues and in vivo mouse pancreatic tumors.

PubMed

Li, Tong; Chen, Hong; Khokhlova, Tatiana; Wang, Yak-Nam; Kreider, Wayne; He, Xuemei; Hwang, Joo Ha

2014-07-01

Pulsed high-intensity focused ultrasound (pHIFU) has been shown to enhance vascular permeability, disrupt tumor barriers and enhance drug penetration into tumor tissue through acoustic cavitation. Monitoring of cavitation activity during pHIFU treatments and knowing the ultrasound pressure levels sufficient to reliably induce cavitation in a given tissue are therefore very important. Here, three metrics of cavitation activity induced by pHIFU and evaluated by confocal passive cavitation detection were introduced: cavitation probability, cavitation persistence and the level of the broadband acoustic emissions. These metrics were used to characterize cavitation activity in several ex vivo tissue types (bovine tongue and liver and porcine adipose tissue and kidney) and gel phantoms (polyacrylamide and agarose) at varying peak-rare factional focal pressures (1-12 MPa) during the following pHIFU protocol: frequency 1.1 MHz, pulse duration 1 ms and pulse repetition frequency 1 Hz. To evaluate the relevance of the measurements in ex vivo tissue, cavitation metrics were also investigated and compared in the ex vivo and in vivo murine pancreatic tumors that develop spontaneously in transgenic KrasLSL.G12 D/+; p53 R172 H/+; PdxCretg/+ (KPC) mice and closely re-capitulate human disease in their morphology. The cavitation threshold, defined at 50% cavitation probability, was found to vary broadly among the investigated tissues (within 2.5-10 MPa), depending mostly on the water-lipid ratio that characterizes the tissue composition. Cavitation persistence and the intensity of broadband emissions depended both on tissue structure and lipid concentration. Both the cavitation threshold and broadband noise emission level were similar between ex vivo and in vivo pancreatic tumor tissue. The largest difference between in vivo and ex vivo settings was found in the pattern of cavitation occurrence throughout pHIFU exposure: it was sporadic in vivo, but it decreased rapidly and stopped

Histological Methods for ex vivo Axon Tracing: A Systematic Review

PubMed Central

Heilingoetter, Cassandra L.; Jensen, Matthew B.

2016-01-01

Objectives Axon tracers provide crucial insight into the development, connectivity, and function of neural pathways. A tracer can be characterized as a substance that allows for the visualization of a neuronal pathway. Axon tracers have previously been used exclusively with in vivo studies; however, newer methods of axon tracing can be applied to ex vivo studies. Ex vivo studies involve the examination of cells or tissues retrieved from an organism. These post mortem methods of axon tracing offer several advantages, such as reaching inaccessible tissues and avoiding survival surgeries. Methods In order to evaluate the quality of the ex vivo tracing methods, we performed a systematic review of various experimental and comparison studies to discern the optimal method of axon tracing. Results The most prominent methods for ex vivo tracing involve enzymatic techniques or various dyes. It appears that there are a variety of techniques and conditions that tend to give better fluorescent character, clarity, and distance traveled in the neuronal pathway. We found direct comparison studies that looked at variables such as the type of tracer, time required, effect of temperature, and presence of calcium, however, there are other variables that have not been compared directly. Discussion We conclude there are a variety of promising tracing methods available depending on the experimental goals of the researcher, however, more direct comparison studies are needed to affirm the optimal method. PMID:27098542

Histological methods for ex vivo axon tracing: A systematic review.

PubMed

Heilingoetter, Cassandra L; Jensen, Matthew B

2016-07-01

Axon tracers provide crucial insight into the development, connectivity, and function of neural pathways. A tracer can be characterized as a substance that allows for the visualization of a neuronal pathway. Axon tracers have previously been used exclusively with in vivo studies; however, newer methods of axon tracing can be applied to ex vivo studies. Ex vivo studies involve the examination of cells or tissues retrieved from an organism. These post mortem methods of axon tracing offer several advantages, such as reaching inaccessible tissues and avoiding survival surgeries. In order to evaluate the quality of the ex vivo tracing methods, we performed a systematic review of various experimental and comparison studies to discern the optimal method of axon tracing. The most prominent methods for ex vivo tracing involve enzymatic techniques or various dyes. It appears that there are a variety of techniques and conditions that tend to give better fluorescent character, clarity, and distance traveled in the neuronal pathway. We found direct comparison studies that looked at variables such as the type of tracer, time required, effect of temperature, and presence of calcium, however, there are other variables that have not been compared directly. We conclude there are a variety of promising tracing methods available depending on the experimental goals of the researcher, however, more direct comparison studies are needed to affirm the optimal method.

A Method for Whole Brain Ex Vivo Magnetic Resonance Imaging with Minimal Susceptibility Artifacts

PubMed Central

Shatil, Anwar S.; Matsuda, Kant M.; Figley, Chase R.

2016-01-01

Magnetic resonance imaging (MRI) is a non-destructive technique that is capable of localizing pathologies and assessing other anatomical features (e.g., tissue volume, microstructure, and white matter connectivity) in postmortem, ex vivo human brains. However, when brains are removed from the skull and cerebrospinal fluid (i.e., their normal in vivo magnetic environment), air bubbles and air–tissue interfaces typically cause magnetic susceptibility artifacts that severely degrade the quality of ex vivo MRI data. In this report, we describe a relatively simple and cost-effective experimental setup for acquiring artifact-free ex vivo brain images using a clinical MRI system with standard hardware. In particular, we outline the necessary steps, from collecting an ex vivo human brain to the MRI scanner setup, and have also described changing the formalin (as might be necessary in longitudinal postmortem studies). Finally, we share some representative ex vivo MRI images that have been acquired using the proposed setup in order to demonstrate the efficacy of this approach. We hope that this protocol will provide both clinicians and researchers with a straight-forward and cost-effective solution for acquiring ex vivo MRI data from whole postmortem human brains. PMID:27965620

An ex vivo investigation into the transurothelial permeability and bladder wall distribution of the nonsteroidal anti-inflammatory ketorolac.

PubMed

Williams, Nicholas A; Bowen, Jenna L; Al-Jayyoussi, Ghaith; Gumbleton, Mark; Allender, Chris J; Li, Jamie; Harrah, Tim; Raja, Aditya; Joshi, Hrishi B

2014-03-03

Transurothelial drug delivery continues to be an attractive treatment option for a range of urological conditions; however, dosing regimens remain largely empirical. Recently, intravesical delivery of the nonsteroidal anti-inflammatory ketorolac has been shown to significantly reduce ureteral stent-related pain. While this latest development provides an opportunity for advancing the management of stent-related pain, clinical translation will undoubtedly require an understanding of the rate and extent of delivery of ketorolac into the bladder wall. Using an ex vivo porcine model, we evaluate the urothelial permeability and bladder wall distribution of ketorolac. The subsequent application of a pharmacokinetic (PK) model enables prediction of concentrations achieved in vivo. Ketorolac was applied to the urothelium and a transurothelial permeability coefficient (Kp) calculated. Relative drug distribution into the bladder wall after 90 min was determined. Ketorolac was able to permeate the urothelium (Kp = 2.63 × 10(-6) cm s(-1)), and after 90 min average concentrations of 400, 141 and 21 μg g(-1) were achieved in the urothelium, lamina propria and detrusor respectively. An average concentration of 87 μg g(-1) was achieved across the whole bladder wall. PK simulations (STELLA) were then carried out, using ex vivo values for Kp and muscle/saline partition coefficient (providing an estimation of vascular clearance), to predict 90 min in vivo ketorolac tissue concentrations. When dilution of the drug solution with urine and vascular clearance were taken into account, a reduced ketorolac concentration of 37 μg g(-1) across the whole bladder wall was predicted. These studies reveal crucial information about the urothelium's permeability to agents such as ketorolac and the concentrations achievable in the bladder wall. It would appear that levels of ketorolac delivered to the bladder wall intravesically would be sufficient to provide an anti-inflammatory effect. The

In Vitro and Ex Vivo Evaluations on Transdermal Delivery of the HIV Inhibitor IQP-0410

PubMed Central

Ham, Anthony S.; Lustig, William; Yang, Lu; Boczar, Ashlee; Buckheit, Karen W.; Buckheit Jr, Robert W.

2013-01-01

The aim of this study was to investigate the physicochemical and in vitro/ex vivo characteristics of the pyrmidinedione IQP-0410 formulated into transdermal films. IQP-0410 is a potent therapeutic anti-HIV nonnucleoside reverse transcriptase inhibitor that would be subjected to extensive first pass metabolism, through conventional oral administration. Therefore, IQP-0410 was formulated into ethyl cellulose/HPMC-based transdermal films via solvent casting. In mano evaluations were performed to evaluate gross physical characteristics. In vitro release studies were performed in both Franz cells and USP-4 dissolution vessels. Ex vivo release and permeability assays were performed on human epidermal tissue models, and the permeated IQP-0410 was collected for in vitro HIV-1 efficacy assays in CEM-SS cells and PBMCs. Film formulation D3 resulted in pliable, strong transdermal films that were loaded with 2% (w/w) IQP-0410. Composed of 60% (w/w) ethyl cellulose and 20% (w/w) HPMC, the films contained < 1.2% (w/w) of water and were hygroscopic resulting in significant swelling under humid conditions. The water permeable nature of the film resulted in complete in vitro dissolution and drug release in 26 hours. When applied to ex vivo epidermal tissues, the films were non-toxic to the tissue and also were non-toxic to HIV target cells used in the in vitro efficacy assays. Over a 3 day application, the films delivered IQP-0410 through the skin tissue at a zero-order rate of 0.94 ± 0.06 µg/cm2/hr with 134 ± 14.7 µM collected in the basal media. The delivered IQP-0410 resulted in in vitro EC50 values against HIV-1 of 2.56 ± 0.40 nM (CEM-SS) and 0.58 ± 0.03 nM (PBMC). The film formulation demonstrated no significant deviation from target values when packaged in foil pouches under standard and accelerated environmental conditions. It was concluded that the transdermal film formulation was a potentially viable method of administering IQP-0410 that warrants further development

Evaluation of transdermal delivery of nanoemulsions in ex vivo porcine skin using two-photon microscopy and confocal laser-scanning microscopy

NASA Astrophysics Data System (ADS)

Choi, Sanghoon; Kim, Jin Woong; Lee, Yong Joong; Delmas, Thomas; Kim, Changhwan; Park, Soyeun; Lee, Ho

2014-10-01

This study experimentally evaluates the self-targeting ability of asiaticoside-loaded nanoemulsions compared with nontargeted nanoemulsions in ex vivo experiments with porcine skin samples. Homebuilt two-photon and confocal laser-scanning microscopes were employed to noninvasively examine the transdermal delivery of two distinct nanoemulsions. Prior to the application of nanoemulsions, we noninvasively observed the morphology of porcine skin using two-photon microscopy. We have successfully visualized the distributions of the targeted and nontargeted nanoemulsions absorbed into the porcine skin samples. Asiaticoside-loaded nanoemulsions showed an improved ex vivo transdermal delivery through the stratum corneum compared with nonloaded nanoemulsions. As a secondary measure, nanoemulsions-applied samples were sliced in the depth direction with a surgical knife in order to obtain the complete depth-direction distribution profile of Nile red fluorescence. XZ images demonstrated that asiaticoside-loaded nanoemulsion penetrated deeper into the skin compared with nontargeted nanoemulsions. The basal layer boundary is clearly visible in the case of the asiaticoside-loaded skin sample. These results reaffirm the feasibility of using self-targeting ligands to improve permeation through the skin barrier for cosmetics and topical drug applications.

In vitro and ex vivo strategies for intracellular delivery

NASA Astrophysics Data System (ADS)

Stewart, Martin P.; Sharei, Armon; Ding, Xiaoyun; Sahay, Gaurav; Langer, Robert; Jensen, Klavs F.

2016-10-01

Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.

Comparison of In Vivo and Ex Vivo MRI for the Detection of Structural Abnormalities in a Mouse Model of Tauopathy

PubMed Central

Holmes, Holly E.; Powell, Nick M.; Ma, Da; Ismail, Ozama; Harrison, Ian F.; Wells, Jack A.; Colgan, Niall; O'Callaghan, James M.; Johnson, Ross A.; Murray, Tracey K.; Ahmed, Zeshan; Heggenes, Morten; Fisher, Alice; Cardoso, M. Jorge; Modat, Marc; O'Neill, Michael J.; Collins, Emily C.; Fisher, Elizabeth M. C.; Ourselin, Sébastien; Lythgoe, Mark F.

2017-01-01

With increasingly large numbers of mouse models of human disease dedicated to MRI studies, compromises between in vivo and ex vivo MRI must be fully understood in order to inform the choice of imaging methodology. We investigate the application of high resolution in vivo and ex vivo MRI, in combination with tensor-based morphometry (TBM), to uncover morphological differences in the rTg4510 mouse model of tauopathy. The rTg4510 mouse also offers a novel paradigm by which the overexpression of mutant tau can be regulated by the administration of doxycycline, providing us with a platform on which to investigate more subtle alterations in morphology with morphometry. Both in vivo and ex vivo MRI allowed the detection of widespread bilateral patterns of atrophy in the rTg4510 mouse brain relative to wild-type controls. Regions of volume loss aligned with neuronal loss and pathological tau accumulation demonstrated by immunohistochemistry. When we sought to investigate more subtle structural alterations in the rTg4510 mice relative to a subset of doxycycline-treated rTg4510 mice, ex vivo imaging enabled the detection of more regions of morphological brain changes. The disadvantages of ex vivo MRI may however mitigate this increase in sensitivity: we observed a 10% global shrinkage in brain volume of the post-mortem tissues due to formalin fixation, which was most notable in the cerebellum and olfactory bulbs. However, many central brain regions were not adversely affected by the fixation protocol, perhaps due to our “in-skull” preparation. The disparity between our TBM findings from in vivo and ex vivo MRI underlines the importance of appropriate study design, given the trade-off between these two imaging approaches. We support the utility of in vivo MRI for morphological phenotyping of mouse models of disease; however, for subtler phenotypes, ex vivo offers enhanced sensitivity to discrete morphological changes. PMID:28408879

Ex vivo validation of photo-magnetic imaging.

PubMed

Luk, Alex; Nouizi, Farouk; Erkol, Hakan; Unlu, Mehmet B; Gulsen, Gultekin

2017-10-15

We recently introduced a new high-resolution diffuse optical imaging technique termed photo-magnetic imaging (PMI), which utilizes magnetic resonance thermometry (MRT) to monitor the 3D temperature distribution induced in a medium illuminated with a near-infrared light. The spatiotemporal temperature distribution due to light absorption can be accurately estimated using a combined photon propagation and heat diffusion model. High-resolution optical absorption images are then obtained by iteratively minimizing the error between the measured and modeled temperature distributions. We have previously demonstrated the feasibility of PMI with experimental studies using tissue simulating agarose phantoms. In this Letter, we present the preliminary ex vivo PMI results obtained with a chicken breast sample. Similarly to the results obtained on phantoms, the reconstructed images reveal that PMI can quantitatively resolve an inclusion with a 3 mm diameter embedded deep in a biological tissue sample with only 10% error. These encouraging results demonstrate the high performance of PMI in ex vivo biological tissue and its potential for in vivo imaging.

In vivo and ex vivo cetuximab sensitivity assay using three-dimensional primary culture system to stratify KRAS mutant colorectal cancer

PubMed Central

Tashiro, Takahiro; Okuyama, Hiroaki; Endo, Hiroko; Kawada, Kenji; Ashida, Yasuko; Ohue, Masayuki; Sakai, Yoshiharu; Inoue, Masahiro

2017-01-01

In clinic, cetuximab, an anti-EGFR antibody, improves treatment outcomes in colorectal cancer (CRC). KRAS-mutant CRC is generally resistant to cetuximab, although difference of the sensitivity among KRAS-mutants has not been studied in detail. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method for cancer cells. We applied CTOS method to investigate whether ex vivo cetuximab sensitivity assays reflect the difference in sensitivity in the xenografts. Firstly, in vivo cetuximab treatment was performed with xenografts derived from 10 CTOS lines (3 KRAS-wildtype and 7 KRAS mutants). All two CTOS lines which exhibited tumor regression were KRAS-wildtype, meanwhile all KRAS-mutant CTOS lines grew more than the initial size: were resistant to cetuximab according to the clinical evaluation criteria, although the sensitivity was quite diverse. We divided KRAS-mutants into two groups; partially responsive group in which cetuximab had a substantial growth inhibitory effect, and resistant group which exhibited no effect. The ex vivo signaling assay with EGF stimulation revealed that the partially responsive group, but not the resistant group, exhibited suppressed ERK phosphorylation ex vivo. Furthermore, two lines from the partially responsive group, but none of the lines in the resistant group, exhibited a combinatory effect of cetuximab and trametinib, a MEK inhibitor, ex vivo and in vivo. Taken together, the results indicate that ex vivo signaling assay reflects the difference in sensitivity in vivo and stratifies KRAS mutant CTOS lines by sensitivity. Therefore, coupling the in vivo and ex vivo assays with CTOS can be a useful platform for understanding the mechanism of diversity in drug sensitivity. PMID:28301591

Modeling Fetal Alcohol Spectrum Disorder: validating an ex vivo primary hippocampal cell culture system

PubMed Central

Tunc-Ozcan, Elif; Ferreira, Adriana B.; Redei, Eva E.

2016-01-01

Background Fetal Alcohol Spectrum Disorder (FASD) is the leading non-genetic cause of mental retardation. There are no treatments for FASD to date. Preclinical in vivo and in vitro studies could help in identifying novel drug targets as for other diseases. Here, we describe an ex vivo model that combines the physiological advantages of prenatal ethanol (E) exposure in vivo with the uniformity of primary fetal hippocampal culture to characterize the effects of prenatal E. The insulin signaling pathways are known to be involved in hippocampal functions. Therefore, we compared the expression of insulin signaling pathway genes between fetal hippocampi (in vivo) and primary hippocampal culture (ex vivo). The similarity of prenatal E effects in these two paradigms would deem the ex vivo culture acceptable to screen possible treatments for FASD. Methods Pregnant Sprague-Dawley rats received one of three diets: ad libitum standard lab chow (control-C), isocaloric pair-fed (PF, nutritional control), and E containing liquid diets from gestational day (GD) 8. Fetal male and female hippocampi were collected either on GD21 (in vivo) or on GD18 for primary culture (ex vivo). Transcript levels of Igf2, Igf2r, Insr, Grb10, Rasgrf1 and Zac1 were measured by RT-qPCR. Results Hippocampal transcript levels differed by prenatal treatment in both males and females with sex differences observed in the expression of Igf2 and Insr. The effect of prenatal E on the hippocampal expression of the insulin pathway genes was parallel in the in vivo and the ex vivo conditions. Conclusions The similarity of gene expression changes in response to prenatal E between the in vivo and the ex vivo conditions ascertain that these effects are already set in the fetal hippocampus at GD18. This strengthens the feasibility of the ex vivo primary hippocampal culture as a tool to test and screen candidate drug targets for FASD. PMID:27162054

Coagulation Management in Jersey Calves: An ex vivo Study.

PubMed

Gröning, Sabine; Maas, Judith; van Geul, Svenja; Rossaint, Rolf; Steinseifer, Ulrich; Grottke, Oliver

2017-01-01

Jersey calves are frequently used as an experimental animal model for in vivo testing of cardiac assist devices or orthopedic implants. In this ex vivo study, we analyzed the coagulation system of the Jersey calves and the potential of human-based coagulation management to circumvent perioperative bleeding complications during surgery. Experimental Procedure: Blood from 7 Jersey calves was subjected to standard laboratory tests and thromboelastometry analysis. An ex vivo model of dilutional coagulopathy was used to study the effects of fibrinogen or prothrombin complex concentrate supplementation. Fibrinolysis was induced with tissue plasminogen activator to identify potential therapeutic strategies involving tranexamic acid or aprotinin. Furthermore, anticoagulation strategies were evaluated by incubating the blood samples with dabigatran or rivaroxaban. Baseline values for thromboelastometry and standard laboratory parameters, including prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin III, and D-dimers, were established. Fifty percent diluted blood showed a statistically significant impairment of hemostasis. The parameters significantly improved after the administration of fibrinogen or prothrombin complex concentrate. Tranexamic acid and aprotinin ameliorated tissue plasminogen activator-induced fibrinolysis. Both dabigatran and rivaroxaban significantly prolonged the coagulation parameters. In this ex vivo study, coagulation factors, factor concentrate, antifibrinolytic reagents, and anticoagulants regularly used in the clinic positively impacted coagulation parameters in Jersey calf blood. © 2017 S. Karger AG, Basel.

Biopharmaceutical evaluation of epigallocatechin gallate-loaded cationic lipid nanoparticles (EGCG-LNs): In vivo, in vitro and ex vivo studies.

PubMed

Fangueiro, Joana F; Calpena, Ana C; Clares, Beatriz; Andreani, Tatiana; Egea, Maria A; Veiga, Francisco J; Garcia, Maria L; Silva, Amélia M; Souto, Eliana B

2016-04-11

Cationic lipid nanoparticles (LNs) have been tested for sustained release and site-specific targeting of epigallocatechin gallate (EGCG), a potential polyphenol with improved pharmacological profile for the treatment of ocular pathologies, such as age-related macular edema, diabetic retinopathy, and inflammatory disorders. Cationic EGCG-LNs were produced by double-emulsion technique; the in vitro release study was performed in a dialysis bag, followed by the drug assay using a previously validated RP-HPLC method. In vitro HET-CAM study was carried out using chicken embryos to determine the potential risk of irritation of the developed formulations. Ex vivo permeation profile was assessed using rabbit cornea and sclera isolated and mounted in Franz diffusion cells. The results show that the use of cationic LNs provides a prolonged EGCG release, following a Boltzmann sigmoidal profile. In addition, EGCG was successfully quantified in both tested ocular tissues, demonstrating the ability of these formulations to reach both anterior and posterior segment of the eye. The pharmacokinetic study of the corneal permeation showed a first order kinetics for both cationic formulations, while EGCG-cetyltrimethylammonium bromide (CTAB) LNs followed a Boltzmann sigmoidal profile and EGCG-dimethyldioctadecylammonium bromide (DDAB) LNs a first order profile. Our studies also proved the safety and non-irritant nature of the developed LNs. Thus, loading EGCG in cationic LNs is recognised as a promising strategy for the treatment of ocular diseases related to anti-oxidant and anti-inflammatory pathways. Copyright © 2016 Elsevier B.V. All rights reserved.

Sex differences in the pro-inflammatory cytokine response to endotoxin unfold in vivo but not ex vivo in healthy humans.

PubMed

Wegner, Alexander; Benson, Sven; Rebernik, Laura; Spreitzer, Ingo; Jäger, Marcus; Schedlowski, Manfred; Elsenbruch, Sigrid; Engler, Harald

2017-07-01

Clinical data indicate that inflammatory responses differ across sexes, but the mechanisms remain elusive. Herein, we assessed in vivo and ex vivo cytokine responses to bacterial endotoxin in healthy men and women to elucidate the role of systemic and cellular factors underlying sex differences in inflammatory responses. Participants received an i.v. injection of low-dose endotoxin (0.4 ng/kg body mass), and plasma TNF-α and IL-6 responses were analyzed over a period of 6 h. In parallel, ex vivo cytokine production was measured in endotoxin-stimulated blood samples obtained immediately before in vivo endotoxin administration. As glucocorticoids (GCs) play an important role in the negative feedback regulation of the inflammatory response, we additionally analyzed plasma cortisol concentrations and ex vivo GC sensitivity of cytokine production. Results revealed greater in vivo pro-inflammatory responses in women compared with men, with significantly higher increases in plasma TNF-α and IL-6 concentrations. In addition, the endotoxin-induced rise in plasma cortisol was more pronounced in women. In contrast, no sex differences in ex vivo cytokine production and GC sensitivity were observed. Together, these findings demonstrate major differences in in vivo and ex vivo responses to endotoxin and underscore the importance of systemic factors underlying sex differences in the inflammatory response.

In vivo visualization and ex vivo quantification of experimental myocardial infarction by indocyanine green fluorescence imaging

PubMed Central

Sonin, Dmitry; Papayan, Garry; Pochkaeva, Evgeniia; Chefu, Svetlana; Minasian, Sarkis; Kurapeev, Dmitry; Vaage, Jarle; Petrishchev, Nickolay; Galagudza, Michael

2016-01-01

The fluorophore indocyanine green accumulates in areas of ischemia-reperfusion injury due to an increase in vascular permeability and extravasation of the dye. The aim of the study was to validate an indocyanine green-based technique of in vivo visualization of myocardial infarction. A further aim was to quantify infarct size ex vivo and compare this technique with the standard triphenyltetrazolium chloride staining. Wistar rats were subjected to regional myocardial ischemia (30 minutes) followed by reperfusion (n = 7). Indocyanine green (0.25 mg/mL in 1 mL of normal saline) was infused intravenously for 10 minutes starting from the 25th minute of ischemia. Video registration in the near-infrared fluorescence was performed. Epicardial fluorescence of indocyanine green corresponded to the injured area after 30 minutes of reperfusion. Infarct size was similar when determined ex vivo using traditional triphenyltetrazolium chloride assay and indocyanine green fluorescent labeling. Intravital visualization of irreversible injury can be done directly by fluorescence on the surface of the heart. This technique may also be an alternative for ex vivo measurements of infarct size. PMID:28101408

In vivo visualization and ex vivo quantification of experimental myocardial infarction by indocyanine green fluorescence imaging.

PubMed

Sonin, Dmitry; Papayan, Garry; Pochkaeva, Evgeniia; Chefu, Svetlana; Minasian, Sarkis; Kurapeev, Dmitry; Vaage, Jarle; Petrishchev, Nickolay; Galagudza, Michael

2017-01-01

The fluorophore indocyanine green accumulates in areas of ischemia-reperfusion injury due to an increase in vascular permeability and extravasation of the dye. The aim of the study was to validate an indocyanine green-based technique of in vivo visualization of myocardial infarction. A further aim was to quantify infarct size ex vivo and compare this technique with the standard triphenyltetrazolium chloride staining. Wistar rats were subjected to regional myocardial ischemia (30 minutes) followed by reperfusion (n = 7). Indocyanine green (0.25 mg/mL in 1 mL of normal saline) was infused intravenously for 10 minutes starting from the 25th minute of ischemia. Video registration in the near-infrared fluorescence was performed. Epicardial fluorescence of indocyanine green corresponded to the injured area after 30 minutes of reperfusion. Infarct size was similar when determined ex vivo using traditional triphenyltetrazolium chloride assay and indocyanine green fluorescent labeling. Intravital visualization of irreversible injury can be done directly by fluorescence on the surface of the heart. This technique may also be an alternative for ex vivo measurements of infarct size.

Systematic Development of Transethosomal Gel System of Piroxicam: Formulation Optimization, In Vitro Evaluation, and Ex Vivo Assessment.

PubMed

Garg, Varun; Singh, Harmanpreet; Bhatia, Amit; Raza, Kaisar; Singh, Sachin Kumar; Singh, Bhupinder; Beg, Sarwar

2017-01-01

Piroxicam is used in the treatment of rheumatoid arthritis, osteoarthritis, and other inflammatory diseases. Upon oral administration, it is reported to cause ulcerative colitis, gastrointestinal irritation, edema and peptic ulcer. Hence, an alternative delivery system has been designed in the form of transethosome. The present study describes the preparation, optimization, characterization, and ex vivo study of piroxicam-loaded transethosomal gel using the central composite design. On the basis of the prescreening study, the concentration of lipids and ethanol was kept in the range of 2-4% w/v and 0-40% v/v, respectively. Formulation was optimized by measuring drug retention in the skin, drug permeation, entrapment efficiency, and vesicle size. Optimized formulation was incorporated in hydrogel and compared with other analogous vesicular (liposomes, ethosomes, and transfersomes) gels for the aforementioned responses. Among the various lipids used, soya phosphatidylcholine (SPL 70) and ethanol in various percentages were found to affect drug retention in the skin, drug permeation, vesicle size, and entrapment efficiency. The optimized batch of transethosome has shown 392.730 μg cm -2 drug retention in the skin, 44.312 μg cm -2  h -1 drug permeation, 68.434% entrapment efficiency, and 655.369 nm vesicle size, respectively. It was observed that the developed transethosomes were found superior in all the responses as compared to other vesicular formulations with improved stability and highest elasticity. Similar observations were noted with its gel formulation.

Rosmarinic Acid Restores Complete Transparency of Sonicated Human Cataract Ex Vivo and Delays Cataract Formation In Vivo.

PubMed

Chemerovski-Glikman, Marina; Mimouni, Michael; Dagan, Yarden; Haj, Esraa; Vainer, Igor; Allon, Raviv; Blumenthal, Eytan Z; Adler-Abramovich, Lihi; Segal, Daniel; Gazit, Ehud; Zayit-Soudry, Shiri

2018-06-19

Cataract, the leading cause of vision impairment worldwide, arises from abnormal aggregation of crystallin lens proteins. Presently, surgical removal is the only therapeutic approach. Recent findings have triggered renewed interest in development of non-surgical treatment alternatives. However, emerging treatments are yet to achieve full and consistent lens clearance. Here, the first ex vivo assay to screen for drug candidates that reduce human lenticular protein aggregation was developed. This assay allowed the identification of two leading compounds as facilitating the restoration of nearly-complete transparency of phacoemulsified cataractous preparation ex vivo. Mechanistic studies demonstrated that both compounds reduce cataract microparticle size and modify their amyloid-like features. In vivo studies confirmed that the lead compound, rosmarinic acid, delays cataract formation and reduces the severity of lens opacification in model rats. Thus, the ex vivo assay may provide an initial platform for broad screening of potential novel therapeutic agents towards pharmacological treatment of cataract.

Effect of Enhancers on in vitro and in vivo Skin Permeation and Deposition of S-Methyl-L-Methionine.

PubMed

Kim, Ki Taek; Kim, Ji Su; Kim, Min-Hwan; Park, Ju-Hwan; Lee, Jae-Young; Lee, WooIn; Min, Kyung Kuk; Song, Min Gyu; Choi, Choon-Young; Kim, Won-Serk; Oh, Hee Kyung; Kim, Dae-Duk

2017-07-01

S-methyl- L -methionine (SMM), also known as vitamin U, is commercially available as skin care cosmetic products for its wound healing and photoprotective effects. However, the low skin permeation expected of SMM due to its hydrophilic nature with a log P value of -3.3, has not been thoroughly addressed. The purpose of this study thus was to evaluate the effect of skin permeation enhancers on the skin permeation/deposition of SMM. Among the enhancers tested for the in vitro skin permeation and deposition of SMM, oleic acid showed the most significant enhancing effect. Moreover, the combination of oleic acid and ethanol further enhanced in vitro permeation and deposition of SMM through hairless mouse skin. Furthermore, the combination of oleic acid and ethanol significantly increased the in vivo deposition of SMM in the epidermis/dermis for 12 hr, which was high enough to exert a therapeutic effect. Therefore, based on the in vitro and in vivo studies, the combination of oleic acid and ethanol was shown to be effective in improving the topical skin delivery of SMM, which may be applied in the cosmetic production process for SMM.

Effect of Enhancers on in vitro and in vivo Skin Permeation and Deposition of S-Methyl-l-Methionine

PubMed Central

Kim, Ki Taek; Kim, Ji Su; Kim, Min-Hwan; Park, Ju-Hwan; Lee, Jae-Young; Lee, WooIn; Min, Kyung Kuk; Song, Min Gyu; Choi, Choon-Young; Kim, Won-Serk; Oh, Hee Kyung; Kim, Dae-Duk

2017-01-01

S-methyl-l-methionine (SMM), also known as vitamin U, is commercially available as skin care cosmetic products for its wound healing and photoprotective effects. However, the low skin permeation expected of SMM due to its hydrophilic nature with a log P value of −3.3, has not been thoroughly addressed. The purpose of this study thus was to evaluate the effect of skin permeation enhancers on the skin permeation/deposition of SMM. Among the enhancers tested for the in vitro skin permeation and deposition of SMM, oleic acid showed the most significant enhancing effect. Moreover, the combination of oleic acid and ethanol further enhanced in vitro permeation and deposition of SMM through hairless mouse skin. Furthermore, the combination of oleic acid and ethanol significantly increased the in vivo deposition of SMM in the epidermis/dermis for 12 hr, which was high enough to exert a therapeutic effect. Therefore, based on the in vitro and in vivo studies, the combination of oleic acid and ethanol was shown to be effective in improving the topical skin delivery of SMM, which may be applied in the cosmetic production process for SMM. PMID:28274096

Glioma survival prediction with the combined analysis of in vivo 11C-MET-PET, ex vivo and patient features by supervised machine learning.

PubMed

Papp, Laszlo; Poetsch, Nina; Grahovac, Marko; Schmidbauer, Victor; Woehrer, Adelheid; Preusser, Matthias; Mitterhauser, Markus; Kiesel, Barbara; Wadsak, Wolfgang; Beyer, Thomas; Hacker, Marcus; Traub-Weidinger, Tatjana

2017-11-24

Gliomas are the most common types of tumors in the brain. While the definite diagnosis is routinely made ex vivo by histopathologic and molecular examination, diagnostic work-up of patients with suspected glioma is mainly done by using magnetic resonance imaging (MRI). Nevertheless, L-S-methyl- 11 C-methionine ( 11 C-MET) Positron Emission Tomography (PET) holds a great potential in characterization of gliomas. The aim of this study was to establish machine learning (ML) driven survival models for glioma built on 11 C-MET-PET, ex vivo and patient characteristics. Methods: 70 patients with a treatment naïve glioma, who had a positive 11 C-MET-PET and histopathology-derived ex vivo feature extraction, such as World Health Organization (WHO) 2007 tumor grade, histology and isocitrate dehydrogenase (IDH1-R132H) mutation status were included. The 11 C-MET-positive primary tumors were delineated semi-automatically on PET images followed by the feature extraction of tumor-to-background ratio based general and higher-order textural features by applying five different binning approaches. In vivo and ex vivo features, as well as patient characteristics (age, weight, height, body-mass-index, Karnofsky-score) were merged to characterize the tumors. Machine learning approaches were utilized to identify relevant in vivo, ex vivo and patient features and their relative weights for 36 months survival prediction. The resulting feature weights were used to establish three predictive models per binning configuration based on a combination of: in vivo/ex vivo and clinical patient information (M36IEP), in vivo and patient-only information (M36IP), and in vivo only (M36I). In addition a binning-independent ex vivo and patient-only (M36EP) model was created. The established models were validated in a Monte Carlo (MC) cross-validation scheme. Results: Most prominent ML-selected and -weighted features were patient and ex vivo based followed by in vivo features. The highest area under the

Correlation of histological and ex-vivo confocal tumor thickness in malignant melanoma.

PubMed

Hartmann, Daniela; Krammer, Sebastian; Ruini, Cristel; Ruzicka, Thomas; von Braunmühl, Tanja

2016-07-01

The ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) is a novel diagnostic method for fresh tissue examination, which has already shown promising results in the evaluation of healthy skin and different skin tumors. In malignant melanoma, the histological tumor thickness plays an essential role for further treatment strategies. The immediate perioperative measurement of tumor thickness by means of ex-vivo CLSM might accelerate the decision for further operating procedures in malignant melanoma. Ten histologically confirmed malignant melanomas from various donor sites were blindly examined by two investigators via ex-vivo CLSM and conventional light microscopy. The histopathological tumor thickness (HTT) and confocal tumor thickness (CTT) were measured independently and evaluated using correlation curves, Spearman's correlation coefficient, and Bland-Altman plots. Bland-Altman plots for HTT and reflectance-mode CTT, as well as for fluorescence-mode CTT, showed high correlations. Spearman's correlation coefficient of HTT and CTT was 1.00 in FM and RM. The mean difference of RM-CTT and FM-CTT versus HTT was 0.09 ± 0.30 mm and 0.19 ± 0.35 mm. In one case, the HTT was identical to the CTT in both modes. This pilot study shows high conformity of CTT and HTT measured in malignant melanoma underlining the potential of ex-vivo CLSM for perioperative decisions on safety margin excisions of malignant melanoma in the future.

Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bager, C.L., E-mail: cba@nordicbioscience.com; Technical University of Denmark; Gudmann, N.

Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art toolsmore » to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFβ and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo

Airway pressure release ventilation during ex vivo lung perfusion attenuates injury.

PubMed

Mehaffey, J Hunter; Charles, Eric J; Sharma, Ashish K; Money, Dustin T; Zhao, Yunge; Stoler, Mark H; Lau, Christine L; Tribble, Curtis G; Laubach, Victor E; Roeser, Mark E; Kron, Irving L

2017-01-01

Critical organ shortages have resulted in ex vivo lung perfusion gaining clinical acceptance for lung evaluation and rehabilitation to expand the use of donation after circulatory death organs for lung transplantation. We hypothesized that an innovative use of airway pressure release ventilation during ex vivo lung perfusion improves lung function after transplantation. Two groups (n = 4 animals/group) of porcine donation after circulatory death donor lungs were procured after hypoxic cardiac arrest and a 2-hour period of warm ischemia, followed by a 4-hour period of ex vivo lung perfusion rehabilitation with standard conventional volume-based ventilation or pressure-based airway pressure release ventilation. Left lungs were subsequently transplanted into recipient animals and reperfused for 4 hours. Blood gases for partial pressure of oxygen/inspired oxygen fraction ratios, airway pressures for calculation of compliance, and percent wet weight gain during ex vivo lung perfusion and reperfusion were measured. Airway pressure release ventilation during ex vivo lung perfusion significantly improved left lung oxygenation at 2 hours (561.5 ± 83.9 mm Hg vs 341.1 ± 136.1 mm Hg) and 4 hours (569.1 ± 18.3 mm Hg vs 463.5 ± 78.4 mm Hg). Likewise, compliance was significantly higher at 2 hours (26.0 ± 5.2 mL/cm H 2 O vs 15.0 ± 4.6 mL/cm H 2 O) and 4 hours (30.6 ± 1.3 mL/cm H 2 O vs 17.7 ± 5.9 mL/cm H 2 O) after transplantation. Finally, airway pressure release ventilation significantly reduced lung edema development on ex vivo lung perfusion on the basis of percentage of weight gain (36.9% ± 14.6% vs 73.9% ± 4.9%). There was no difference in additional edema accumulation 4 hours after reperfusion. Pressure-directed airway pressure release ventilation strategy during ex vivo lung perfusion improves the rehabilitation of severely injured donation after circulatory death lungs. After transplant, these lungs demonstrate

Terbinafine Hydrochloride Trans-ungual Delivery via Nanovesicular Systems: In Vitro Characterization and Ex Vivo Evaluation.

PubMed

Elsherif, Noha Ibrahim; Shamma, Rehab Nabil; Abdelbary, Ghada

2017-02-01

Treating a nail infection like onychomycosis is challenging as the human nail plate acts as a formidable barrier against all drug permeation. Available oral and topical treatments have several setbacks. Terbinafine hydrochloride (TBH), belonging to the allylamine class, is mainly used for treatment of onychomycosis. This study aims to formulate TBH in a nanobased spanlastic vesicular carrier that enables and enhances the drug delivery through the nail. The nanovesicles were formulated by ethanol injection method, using either Span® 60 or Span® 65, together with Tween 80 or sodium deoxycholate as an edge activator. A full factorial design was implemented to study the effect of different formulation and process variables on the prepared TBH-loaded spanlastic nanovesicles. TBH entrapment efficiency percentages, particle size diameter, percentage drug released after 2 h and 8 h were selected as dependent variables. Optimization was performed using Design-Expert® software to obtain an optimized formulation with high entrapment efficiency (62.35 ± 8.91%), average particle size of 438.45 ± 70.5 nm, and 29.57 ± 0.93 and 59.53 ± 1.73% TBH released after 2 and 8 h, respectively. The optimized formula was evaluated using differential scanning calorimetry and X-ray diffraction and was also morphologically examined using transmission electron microscopy. An ex vivo study was conducted to determine the permeation and retainment of the optimized formulation in a human cadaver nail plate, and confocal laser scanning microscope was used to show the extent of formulation permeation. In conclusion, the results confirmed that spanlastics exhibit promising results for the trans-ungual delivery of TBH.

Impacts of chemical enhancers on skin permeation and deposition of terbinafine.

PubMed

Erdal, Meryem Sedef; Peköz, Ayca Yıldız; Aksu, Buket; Araman, Ahmet

2014-08-01

The addition of chemical enhancers into formulations is the most commonly employed approach to overcome the skin barrier. The objective of this work was to evaluate the effect of vehicle and chemical enhancers on the skin permeation and accumulation of terbinafine, an allylamine antifungal drug. Terbinafine (1% w/w) was formulated as a Carbopol 934 P gel formulation in presence and absence of three chemical enhancers, nerolidol, dl-limonene and urea. Terbinafine distribution and deposition in stratum corneum (SC) and skin following 8-h ex vivo permeation study was determined using a sequential tape stripping procedure. The conformational order of SC lipids was investigated by ATR-FTIR spectroscopy. Nerolidol containing gel formulation produced significantly higher enhancement in terbinafine permeation through skin and its skin accumulation was increased. ATR-FTIR results showed enhancer induced lipid bilayer disruption in SC. Urea resulted in enhanced permeation of terbinafine across the skin and a balanced distribution to the SC was achieved. But, dl-limonene could not minimize the accumulation of terbinafine in the upper SC. Nerolidol dramatically improved the skin permeation and deposition of terbinafine in the skin that might help to optimize targeting of the drug to the epidermal sites as required for both of superficial and deep cutaneous fungal infections.

Ex vivo tracheomalacia model with 3D-printed external tracheal splint.

PubMed

Kaye, Rachel; Goldstein, Todd; Aronowitz, Danielle; Grande, Daniel A; Zeltsman, David; Smith, Lee P

2017-04-01

To design and evaluate an ex vivo model of tracheomalacia with and without a three-dimensional (3D)-printed external tracheal splint. Prospective, ex vivo animal trial. Three groups of ex vivo porcine tracheas were used: 1) control (unmanipulated trachea), 2) tracheomalacia (tracheal rings partially incised and crushed), and 3) splinted tracheomalacia (external custom tracheal splint fitted onto group 2 trachea). Each end of an ex vivo trachea was sealed with a custom-designed and 3D-printed cap; a transducer was placed through one end to measure the pressure inside the trachea. Although the negative pressure was applied to the tracheal lumen, the tracheal wall collapse was measured externally and internally using a bronchoscope. Each group had at least three recorded trials. Tracheal diameter was evaluated using ImageJ software (National Institutes of Health, Bethesda, MD) and was averaged between two raters. Average tracheal occlusion percentage was compared using Student t test. The average occlusion was 31% for group 1, 87.4% for group 2, and 20% for group 3. Significant differences were found between the control and tracheomalacia groups (P < 0.01) and the tracheomalacia and splinted tracheomalacia groups (P < 0.01). There was no significant difference between the control and splinted tracheomalacia groups (P = 0.13). Applied pressure was plotted against occlusion and regression line slope differed between the tracheomalacia (0.91) and control (0.12) or splinted tracheomalacia (0.39) groups. We demonstrate the potential for an ex vivo tracheomalacia model to reproduce airway collapse and show that this collapse can be treated successfully with a 3D-printed external splint. These results are promising and justify further studies. N/A. Laryngoscope, 127:950-955, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo.

PubMed

Esakky, Prabagaran; Hansen, Deborah A; Drury, Andrea M; Felder, Paul; Cusumano, Andrew; Moley, Kelle H

2018-01-01

Male exposure to cigarette smoke is associated with seminal defects and with congenital anomalies and childhood cancers in offspring. In mice, paternal exposure to cigarette smoke condensate (CSC) causes molecular defects in germ cells and phenotypic effects in their offspring. Here we used an ex vivo testicular explant model and in vivo exposure to determine the concentration at which CSC impairs spermatogenesis and offspring development. We explanted testis tissue at postnatal day (P)5.5 and cultured it until P11.5. Assessment of growth parameters by analyzing expression of cell-specific markers revealed that the explant system maintained structural and functional integrity. We exposed the P5.5 to -11.5 explants to various concentrations (40-160 µg/ml) of CSC and confirmed that nicotine in the CSC was metabolized to cotinine. We assessed various growth and differentiation parameters, as well as testosterone production, and observed that many spermatogenesis features were impaired at 160 µg/ml CSC. The same parameters were impaired by a similar CSC concentration in vivo Finally, females mated to males that were exposed to 160 µg/ml CSC neonatally had increased rates of pup resorption. We conclude that male exposure to CSC impairs offspring development and that the concentration at which CSC impairs spermatogenesis is similar in vivo and ex vivo. Given that the concentrations of CSC we used contained similar doses of nicotine as human smokers are exposed to, we argue that our model mimics human male reproductive effects of smoking.-Esakky, P., Hansen, D. A., Drury, A. M., Felder, P., Cusumano, A., Moley, K. H. Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo . © FASEB.

Ebola Virus Persistence in Semen Ex Vivo.

PubMed

Fischer, Robert J; Judson, Seth; Miazgowicz, Kerri; Bushmaker, Trent; Munster, Vincent J

2016-02-01

On March 20, 2015, a case of Ebola virus disease was identified in Liberia that most likely was transmitted through sexual contact. We assessed the efficiency of detecting Ebola virus in semen samples by molecular diagnostics and the stability of Ebola virus in ex vivo semen under simulated tropical conditions.

Steps for the autologous ex vivo perfused porcine liver-kidney experiment.

PubMed

Chung, Wen Yuan; Eltweri, Amar M; Isherwood, John; Haqq, Jonathan; Ong, Seok Ling; Gravante, Gianpiero; Lloyd, David M; Metcalfe, Matthew S; Dennison, Ashley R

2013-12-18

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

High embryonic recovery rates with in vivo and ex vivo techniques in the bitch.

PubMed

Luz, M R; de Holanda, C C; Pereira, J J; Freitas, P M C; Salgado, A E P; Giannotti, J Di Giorgio; de Oliveira, S B; Teixeira, N S; Guaitolini, C R de Freitas

2011-08-01

The embryonic collection techniques in dogs present a vast methodological variation and low recovery rates. The objectives were to compare and describe two techniques as to the recovery of canine embryos, on the 12th day after the first mating or artificial insemination. Embryos were recovered through uterine horn flushing in vivo, before performing the ovariohysterectomy (OHE) (Group 1; n = 9) or ex vivo, immediately after the OHE (Group 2; n = 9). In total, 43 and 47 embryonic structures were recovered in Groups 1 and 2, respectively. There was no significant difference (p > 0.05) between groups on recovery rates (72.8% and 81.0%, respectively). We inferred that both in vivo and ex vivo techniques allow a high rate of embryonic recovery; in the collection technique prior to the OHE, it is essential to carefully handle the reproductive system during the trans-surgical period and that the 12th day (D12) after the first mating/artificial insemination is an efficient option for the high recovery rate of morulae and blastocysts. © 2010 Blackwell Verlag GmbH.

Evaluation of carotenoids and reactive oxygen species in human skin after UV irradiation: a critical comparison between in vivo and ex vivo investigations.

PubMed

Meinke, Martina C; Müller, Robert; Bechtel, Anne; Haag, Stefan F; Darvin, Maxim E; Lohan, Silke B; Ismaeel, Fakher; Lademann, Jürgen

2015-03-01

UV irradiation is one of the most harmful exogenous factors for the human skin. In addition to the development of erythema, free radicals, that is reactive oxygen species (ROS), are induced under its influence and promote the development of oxidative stress in the skin. Several techniques are available for determining the effect of UV irradiation. Resonance Raman spectroscopy (RRS) measures the reduction of the carotenoid concentration, while electron paramagnetic resonance (EPR) spectroscopy enables the analysis of the production of free radicals. Depending on the method, the skin parameters are analysed in vivo or ex vivo. This study provides a critical comparison between in vivo and ex vivo investigations on the ROS formation and carotenoid depletion caused by UV irradiation in human skin. The oxygen content of tissue was also determined. It was shown that the antioxidant status measured in the skin samples in vivo and ex vivo was different. The depletion in the carotenoid concentration in vivo exceeded the value determined ex vivo by a factor of about 1.5, and the radical formation after UV irradiation was significantly greater in vivo by a factor of 3.5 than that measured in excised human skin, which can be explained by the lack of oxygen ex vivo. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Caspase inhibition supports proper gene expression in ex vivo mouse limb cultures.

PubMed

De Valck, D; Luyten, F P

2001-10-01

We standardized conditions for ex vivo mouse limb culture to study cartilage maturation and joint formation. We compared 12.5 d.p.c. mouse forelimbs that were cultured either mounted or freely rotating for up to 72 h. Limb outgrowth progressed ex vivo at a variable rate as compared to its development in vivo, spanning approximately 48 h. Although cartilage maturation and joint formation developed grossly normal, aberrant expression of skeletal marker genes was seen. Interestingly, no regression of the interdigital webs took place in mounted cultures, in contrast to limited webbing under freely rotating conditions. Caspase inhibition, by addition of zVAD-fmk to the culture medium of freely rotating limbs, supported proper gene expression associated with skeletal development, and prevented interdigital web regression. Taken together, a freely rotating ex vivo culture for mouse limb outgrowth that is combined with caspase inhibition provides a good model to study cartilage maturation and joint formation.

Transethosomal gels as carriers for the transdermal delivery of colchicine: statistical optimization, characterization, and ex vivo evaluation

PubMed Central

Abdulbaqi, Ibrahim M; Darwis, Yusrida; Assi, Reem Abou; Khan, Nurzalina Abdul Karim

2018-01-01

Introduction Colchicine is used for the treatment of gout, pseudo-gout, familial Mediterranean fever, and many other illnesses. Its oral administration is associated with poor bioavailability and severe gastrointestinal side effects. The drug is also known to have a low therapeutic index. Thus to overcome these drawbacks, the transdermal delivery of colchicine was investigated using transethosomal gels as potential carriers. Methods Colchicine-loaded transethosomes (TEs) were prepared by the cold method and statistically optimized using three sets of 24 factorial design experiments. The optimized formulations were incorporated into Carbopol 940® gel base. The prepared colchicine-loaded transethosomal gels were further characterized for vesicular size, dispersity, zeta potential, drug content, pH, viscosity, yield, rheological behavior, and ex vivo skin permeation through Sprague Dawley rats’ back skin. Results The results showed that the colchicine-loaded TEs had aspherical irregular shape, nanometric size range, and high entrapment efficiency. All the formulated gels exhibited non-Newtonian plastic flow without thixotropy. Colchicine-loaded transethosomal gels were able to significantly enhance the skin permeation parameters of the drug in comparison to the non-ethosomal gel. Conclusion These findings suggested that the transethosomal gels are promising carriers for the transdermal delivery of colchicine, providing an alternative route for drug administration. PMID:29670336

Transethosomal gels as carriers for the transdermal delivery of colchicine: statistical optimization, characterization, and ex vivo evaluation.

PubMed

Abdulbaqi, Ibrahim M; Darwis, Yusrida; Assi, Reem Abou; Khan, Nurzalina Abdul Karim

2018-01-01

Colchicine is used for the treatment of gout, pseudo-gout, familial Mediterranean fever, and many other illnesses. Its oral administration is associated with poor bioavailability and severe gastrointestinal side effects. The drug is also known to have a low therapeutic index. Thus to overcome these drawbacks, the transdermal delivery of colchicine was investigated using transethosomal gels as potential carriers. Colchicine-loaded transethosomes (TEs) were prepared by the cold method and statistically optimized using three sets of 24 factorial design experiments. The optimized formulations were incorporated into Carbopol 940 ® gel base. The prepared colchicine-loaded transethosomal gels were further characterized for vesicular size, dispersity, zeta potential, drug content, pH, viscosity, yield, rheological behavior, and ex vivo skin permeation through Sprague Dawley rats' back skin. The results showed that the colchicine-loaded TEs had aspherical irregular shape, nanometric size range, and high entrapment efficiency. All the formulated gels exhibited non-Newtonian plastic flow without thixotropy. Colchicine-loaded transethosomal gels were able to significantly enhance the skin permeation parameters of the drug in comparison to the non-ethosomal gel. These findings suggested that the transethosomal gels are promising carriers for the transdermal delivery of colchicine, providing an alternative route for drug administration.

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Tissue culture media for human ex vivo tissue and... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Tissue culture media for human ex vivo tissue and... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Tissue culture media for human ex vivo tissue and... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

Development of an ex vivo cellular model of rheumatoid arthritis: critical role of CD14-positive monocyte/macrophages in the development of pannus tissue.

PubMed

Nozaki, Toshiko; Takahashi, Kyoko; Ishii, Osamu; Endo, Sachio; Hioki, Kyoji; Mori, Toshihito; Kikukawa, Tadahiro; Boumpas, Dimitrios T; Ozaki, Shoichi; Yamada, Hidehiro

2007-09-01

To establish an ex vivo cellular model of pannus, the aberrant overgrowth of human synovial tissue (ST). Inflammatory cells that infiltrated pannus tissue from patients with rheumatoid arthritis (RA) were collected without enzyme digestion, and designated as ST-derived inflammatory cells. Single-cell suspensions of ST-derived inflammatory cells were cultured in medium alone. Levels of cytokines produced in culture supernatants were measured using enzyme-linked immunosorbent assay kits. ST-derived inflammatory cells were transferred into the joints of immunodeficient mice to explore whether these cells could develop pannus. CD14 and CD2 cells were depleted by negative selection. Culture of ST-derived inflammatory cells from 92 of 111 patients with RA resulted in spontaneous reconstruction of inflammatory tissue in vitro within 4 weeks. Ex vivo tissue contained fibroblasts, macrophages, T cells, and tartrate-resistant acid phosphatase-positive multinucleated cells. On calcium phosphate-coated slides, ST-derived inflammatory cell cultures showed numerous resorption pits. ST-derived inflammatory cell cultures continuously produced matrix metalloproteinase 9 and proinflammatory cytokines associated with osteoclastogenesis, such as tumor necrosis factor alpha, interleukin-8, and macrophage colony-stimulating factor. More importantly, transferring ST-derived inflammatory cells into the joints of immunodeficient mice resulted in the development of pannus tissue and erosive joint lesions. Both in vitro development and in vivo development of pannus tissue by ST-derived inflammatory cells were inhibited by depleting CD14-positive, but not CD2-positive, cells from ST-derived inflammatory cells. These findings suggest that overgrowth of inflammatory cells from human rheumatoid synovium simulates the development of pannus. This may prove informative in the screening of potential antirheumatic drugs.

Dissolution and dissolution/permeation experiments for predicting systemic exposure following oral administration of the BCS class II drug clarithromycin.

PubMed

Kristin, Forner; René, Holm; Boontida, Morakul; Buraphacheep, Junyaprasert Varaporn; Maximilian, Ackermann; Johanna, Mazur; Peter, Langguth

2017-04-01

In order to save time and resources in early drug development, in vitro methods that correctly predict the formulation effect on oral drug absorption are necessary. The aim of this study was to 1) evaluate various BCS class II drug formulations with in vitro methods and in vivo in order to 2) determine which in vitro method best correlates with the in vivo results. Clarithromycin served as model compound in formulations with different particle sizes and content of excipients. The performed in vitro experiments were dissolution and dissolution/permeation experiments across two types of membrane, Caco-2 cells and excised rat intestinal sheets. The in vivo study was performed in rats. The oral absorption was enhanced by downsizing drug particles and by increasing the excipient concentration. This correlated strongly with the flux across Caco-2 cells but not with the other in vitro experiments. The insufficient correlation with the dissolution experiments can be partly explained by excipient caused problems during the filtration step. The very poor correlation of the in vivo data with the flux across excised rat intestinal sheets might be due to an artificially enlarged mucus layer ex vivo. In conclusion, downsizing BCS class II drug particles and the addition of surfactants enhanced the in vivo absorption, which was best depicted by dissolution/permeation experiments across Caco-2 cells. This setup is proposed as best model to predict the in vivo formulation effect. Also, this is the first study to evaluate the impact of the nature of the permeation membrane in dissolution/permeation experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

Effectiveness of hand washing on the removal of iron oxide nanoparticles from human skin ex vivo.

PubMed

Lewinski, Nastassja A; Berthet, Aurélie; Maurizi, Lionel; Eisenbeis, Antoine; Hopf, Nancy B

2017-08-01

In this study, the effectiveness of washing with soap and water in removing nanoparticles from exposed skin was investigated. Dry, nanoscale hematite (α-Fe 2 O 3 ) or maghemite (γ-Fe 2 O 3 ) powder, with primary particle diameters between 20-30 nm, were applied to two samples each of fresh and frozen ex vivo human skin in two independent experiments. The permeation of nanoparticles through skin, and the removal of nanoparticles after washing with soap and water were investigated. Bare iron oxide nanoparticles remained primarily on the surface of the skin, without penetrating beyond the stratum corneum. Skin exposed to iron oxide nanoparticles for 1 and 20 hr resulted in removal of 85% and 90%, respectively, of the original dose after washing. In the event of dermal exposure to chemicals, removal is essential to avoid potential local irritation or permeation across skin. Although manufactured at an industrial scale and used extensively in laboratory experiments, limited data are available on the removal of engineered nanoparticles after skin contact. Our finding raises questions about the potential consequences of nanoparticles remaining on the skin and whether alternative washing methods should be proposed. Further studies on skin decontamination beyond use of soap and water are needed to improve the understanding of the potential health consequences of dermal exposure to nanoparticles.

The use of ex vivo human skin tissue for genotoxicity testing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reus, Astrid A.; Usta, Mustafa; Krul, Cyrille A.M., E-mail: cyrille.krul@tno.nl

2012-06-01

As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positivemore » or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air–liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. -- Highlights: ► We use human skin obtained from surgery for genotoxicity evaluation of chemicals. ► We use the comet assay as parameter for genotoxicity in ex vivo human skin. ► Sensitivity, specificity and accuracy to predict in vivo genotoxins are determined. ► Sensitivity, specificity and accuracy are 89%, 90% and 90%, respectively. ► The

Photodynamic antimicrobial effect of safranine O on an ex vivo periodontal biofilm.

PubMed

Voos, Amelia C; Kranz, Stefan; Tonndorf-Martini, Silke; Voelpel, Andrea; Sigusch, Holger; Staudte, Henrike; Albrecht, Volker; Sigusch, Bernd W

2014-03-01

The increasing resistance of oral pathogens against antibiotic measures urgently requires new therapeutic strategies. In this context, antimicrobial photodynamic therapy (aPDT) may play a crucial part in the future. The aim of the present study was to compare the antibacterial efficiency of aPDT using the photosensitizer safranine O with that of chlorhexidine (0.2% CHX) on an ex vivo biofilm. First the antibacterial activity of both measures against planktonic cultures of Streptococcus gordonii ATCC 33399, Streptococcus mutans ATCC 25175, Fusobacterium nucleatum ATCC 10953, Aggregatibacter actinomycetemcomitans ATCC 33384 and Porphyromonas gingivalis ATCC 33277 was observed. Then a patient specific ex vivo biofilm was established from plaque and saliva samples of patients (n = 19) with chronic periodontitis. The antibacterial effects of aPDT and of 0.2% CHX were determined on the ex vivo biofilms cultivated for 24 and 72 hours. After cultivation of the treated samples on blood agar (2 days) the results were quantified by counting the colony forming units (cfu/ml). Photodynamic treatment with safranine O showed a distinct antibacterial effect on F. nucleatum and P. gingivalis. Whereas S. gordonii was suppressed completely by aPDT, treatment with 0.2% CHX caused only a partial reduction. In the ex vivo biofilm model (24-hour biofilm), aPDT caused a significantly higher bacterial killing than treatment with 0.2% CHX. Compared to the untreated control, there was no significant difference on the 72-hour biofilm for both methods. The results show that oral-pathogenic species in planktonic solution can be suppressed significantly by aPDT with safranine O. Especially for bacteria in a 24-hour ex vivo biofilm, this method is more effective than treatment with 0.2% CHX. Both antibacterial treatments did not show any significant effect on the biofilm cultivated for 72 hours. © 2014 Wiley Periodicals, Inc.

Contrast-enhanced CT with a High-Affinity Cationic Contrast Agent for Imaging ex Vivo Bovine, Intact ex Vivo Rabbit, and in Vivo Rabbit Cartilage

PubMed Central

Stewart, Rachel C.; Bansal, Prashant N.; Entezari, Vahid; Lusic, Hrvoje; Nazarian, Rosalynn M.; Snyder, Brian D.

2013-01-01

Purpose: To quantify the affinity of a cationic computed tomography (CT) contrast agent (CA4+) and that of an anionic contrast agent (ioxaglate) to glycosaminoglycans (GAGs) in ex vivo cartilage tissue explants and to characterize the in vivo diffusion kinetics of CA4+ and ioxaglate in a rabbit model. Materials and Methods: All in vivo procedures were approved by the institutional animal care and use committee. The affinities of ioxaglate and CA4+ to GAGs in cartilage (six bovine osteochondral plugs) were quantified by means of a modified binding assay using micro-CT after plug equilibration in serial dilutions of each agent. The contrast agents were administered intraarticularly to the knee joints of five New Zealand white rabbits to determine the in vivo diffusion kinetics and cartilage tissue imaging capabilities. Kinetics of diffusion into the femoral groove cartilage and relative contrast agent uptake into bovine plugs were characterized by means of nonlinear mixed-effects models. Diffusion time constants (τ) were compared by using a Student t test. Results: The uptake of CA4+ in cartilage was consistently over 100% of the reservoir concentration, whereas it was only 59% for ioxaglate. In vivo, the contrast material–enhanced cartilage reached a steady CT attenuation for both CA4+ and ioxaglate, with τ values of 13.8 and 6.5 minutes, respectively (P = .04). The cartilage was easily distinguishable from the surrounding tissues for CA4+ (12 mg of iodine per milliliter); comparatively, the anionic contrast agent provided less favorable imaging results, even when a higher concentration was used (80 mg of iodine per milliliter). Conclusion: The affinity of the cationic contrast agent CA4+ to GAGs enables high-quality imaging and segmentation of ex vivo bovine and rabbit cartilage, as well as in vivo rabbit cartilage. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112246/-/DC1 PMID:23192774

Multiparameter comparative analysis reveals differential impacts of various cytokines on CART cell phenotype and function ex vivo and in vivo

PubMed Central

Xu, Xiao-Jun; Song, De-Gang; Poussin, Mathilde; Ye, Qunrui; Sharma, Prannda; Rodríguez-García, Alba; Tang, Yong-Min; Powell, Daniel J.

2016-01-01

Exogenous cytokines are widely applied to enhance the anti-tumor ability of immune cells. However, systematic comparative studies of their effects on chimeric antigen receptor (CAR)-engineered T (CART) cells are lacking. In this study, CART cells targeting folate receptor-alpha were generated and expanded ex vivo in the presence of different cytokines (IL-2, IL-7, IL-15, IL-18, and IL-21), and their expansion, phenotype and cytotoxic capacity were evaluated, in vitro and in vivo. Moreover, the effect of the administration of these cytokines along with CART cells in vivo was also studied. IL-2, IL-7, and IL-15 favored the ex vivo expansion of CART cells compared to other cytokines or no cytokine treatment. IL-7 induced the highest proportion of memory stem cell-like CART cells in the final product, and IL-21 supported the expansion of CART cells with a younger phenotype, while IL-2 induced more differentiated CART cells. IL-2 and IL-15-exposed CART cells secreted more proinflammatory cytokines and presented stronger tumor-lysis ability in vitro. However, when tested in vivo, CART cells exposed to IL-2 ex vivo showed the least anti-tumor effect. In contrast, the administration of IL-15 and IL-21 in combination with CART cells in vivo increased their tumor killing capacity. According to our results, IL-7 and IL-15 show promise to promote ex vivo expansion of CART cells, while IL-15 and IL-21 seem better suited for in vivo administration after CART cell infusion. Collectively, these results may have a profound impact on the efficacy of CART cells in both hematologic and solid cancers. PMID:27409425

Ex vivo and in vivo label-free imaging of lymphatic vessels using OCT lymphangiography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Gong, Peijun; Es'haghian, Shaghayegh; Karnowski, Karol; Rea, Suzanne; Wood, Fiona M.; Yu, Dao-Yi; McLaughlin, Robert A.; Sampson, David D.

2017-02-01

We have been developing an automated method to image lymphatic vessels both ex vivo and in vivo with optical coherence tomography (OCT), using their optical transparency. Our method compensates for the OCT signal attenuation for each A-scan in combination with the correction of the confocal function and sensitivity fall-off, enabling reliable thresholding of lymphatic vessels from the OCT scans. Morphological image processing with a segment-joining algorithm is also incorporated into the method to mitigate partial-volume artifacts, which are particularly evident with small lymphatic vessels. Our method is demonstrated for two different clinical application goals: the monitoring of conjunctival lymphatics for surgical guidance and assessment of glaucoma treatment; and the longitudinal monitoring of human burn scars undergoing laser ablation treatment. We present examples of OCT lymphangiography ex vivo on porcine conjunctivas and in vivo on human burn scars, showing the visualization of the lymphatic vessel network and their longitudinal changes due to treatment.

Comparative lung toxicity of engineered nanomaterials utilizing in vitro, ex vivo and in vivo approaches.

PubMed

Kim, Yong Ho; Boykin, Elizabeth; Stevens, Tina; Lavrich, Katelyn; Gilmour, M Ian

2014-11-26

Although engineered nanomaterials (ENM) are currently regulated either in the context of a new chemical, or as a new use of an existing chemical, hazard assessment is still to a large extent reliant on information from historical toxicity studies of the parent compound, and may not take into account special properties related to the small size and high surface area of ENM. While it is important to properly screen and predict the potential toxicity of ENM, there is also concern that current toxicity tests will require even heavier use of experimental animals, and reliable alternatives should be developed and validated. Here we assessed the comparative respiratory toxicity of ENM in three different methods which employed in vivo, in vitro and ex vivo toxicity testing approaches. Toxicity of five ENM (SiO2 (10), CeO2 (23), CeO2 (88), TiO2 (10), and TiO2 (200); parentheses indicate average ENM diameter in nm) were tested in this study. CD-1 mice were exposed to the ENM by oropharyngeal aspiration at a dose of 100 μg. Mouse lung tissue slices and alveolar macrophages were also exposed to the ENM at concentrations of 22-132 and 3.1-100 μg/mL, respectively. Biomarkers of lung injury and inflammation were assessed at 4 and/or 24 hr post-exposure. Small-sized ENM (SiO2 (10), CeO2 (23), but not TiO2 (10)) significantly elicited pro-inflammatory responses in mice (in vivo), suggesting that the observed toxicity in the lungs was dependent on size and chemical composition. Similarly, SiO2 (10) and/or CeO2 (23) were also more toxic in the lung tissue slices (ex vivo) and alveolar macrophages (in vitro) compared to other ENM. A similar pattern of inflammatory response (e.g., interleukin-6) was observed in both ex vivo and in vitro when a dose metric based on cell surface area (μg/cm(2)), but not culture medium volume (μg/mL) was employed. Exposure to ENM induced acute lung inflammatory effects in a size- and chemical composition-dependent manner. The cell culture and lung

Free radicals induced by sunlight in different spectral regions - in vivo versus ex vivo study.

PubMed

Lohan, Silke B; Müller, Robert; Albrecht, Stephanie; Mink, Kathrin; Tscherch, Kathrin; Ismaeel, Fakher; Lademann, Jürgen; Rohn, Sascha; Meinke, Martina C

2016-05-01

Sunlight represents an exogenous factor stimulating formation of free radicals which can induce cell damage. To assess the effect of the different spectral solar regions on the development of free radicals in skin, in vivo electron paramagnetic resonance (EPR) investigations with human volunteers and ex vivo studies on excised human and porcine skin were carried out. For all skin probes, the ultraviolet (UV) spectral region stimulates the most intensive radical formation, followed by the visible (VIS) and the near infrared (NIR) regions. A comparison between the different skin models shows that for UV light, the fastest and highest production of free radicals could be detected in vivo, followed by excised porcine and human skin. The same distribution pattern was found for the VIS/NIR spectral regions, whereby the differences in radical formation between in vivo and ex vivo were less pronounced. An analysis of lipid composition in vivo before and after exposure to UV light clearly showed modifications in several skin lipid components; a decrease of ceramide subclass [AP2] and an increase of ceramide subclass [NP2], sodium cholesterol sulphate and squalene (SQ) were detectable. In contrast, VIS/NIR irradiation led to an increase of ceramides [AP2] and SCS, and a decrease of SQ. These results, which are largely comparable for the different skin models investigated in vivo and ex vivo, indicate that radiation exposure in different spectral regions strongly influences radical production in skin and also results in changes in skin lipid composition, which is essential for barrier function. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ex vivo diffusion MRI of the human brain: Technical challenges and recent advances.

PubMed

Roebroeck, Alard; Miller, Karla L; Aggarwal, Manisha

2018-06-04

This review discusses ex vivo diffusion magnetic resonance imaging (dMRI) as an important research tool for neuroanatomical investigations and the validation of in vivo dMRI techniques, with a focus on the human brain. We review the challenges posed by the properties of post-mortem tissue, and discuss state-of-the-art tissue preparation methods and recent advances in pulse sequences and acquisition techniques to tackle these. We then review recent ex vivo dMRI studies of the human brain, highlighting the validation of white matter orientation estimates and the atlasing and mapping of large subcortical structures. We also give particular emphasis to the delineation of layered gray matter structure with ex vivo dMRI, as this application illustrates the strength of its mesoscale resolution over large fields of view. We end with a discussion and outlook on future and potential directions of the field. © 2018 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

Comparison of in vivo vs. ex situ obtained material properties of sheep common carotid artery.

PubMed

Smoljkić, Marija; Verbrugghe, Peter; Larsson, Matilda; Widman, Erik; Fehervary, Heleen; D'hooge, Jan; Vander Sloten, Jos; Famaey, Nele

2018-05-01

Patient-specific biomechanical modelling can improve preoperative surgical planning. This requires patient-specific geometry as well as patient-specific material properties as input. The latter are, however, still quite challenging to estimate in vivo. This study focuses on the estimation of the mechanical properties of the arterial wall. Firstly, in vivo pressure, diameter and thickness of the arterial wall were acquired for sheep common carotid arteries. Next, the animals were sacrificed and the tissue was stored for mechanical testing. Planar biaxial tests were performed to obtain experimental stress-stretch curves. Finally, parameters for the hyperelastic Mooney-Rivlin and Gasser-Ogden-Holzapfel (GOH) material model were estimated based on the in vivo obtained pressure-diameter data as well as on the ex situ experimental stress-stretch curves. Both material models were able to capture the in vivo behaviour of the tissue. However, in the ex situ case only the GOH model provided satisfactory results. When comparing different fitting approaches, in vivo vs. ex situ, each of them showed its own advantages and disadvantages. The in vivo approach estimates the properties of the tissue in its physiological state while the ex situ approach allows to apply different loadings to properly capture the anisotropy of the tissue. Both of them could be further enhanced by improving the estimation of the stress-free state, i.e. by adding residual circumferential stresses in vivo and by accounting for the flattening effect of the tested samples ex vivo. • Competing interests: none declared • Word count: 4716. Copyright © 2018. Published by Elsevier Ltd.

Ex vivo blood vessel bioreactor for analysis of the biodegradation of magnesium stent models with and without vessel wall integration

PubMed Central

Wang, Juan; Liu, Lumei; Wu, Yifan; Maitz, Manfred F.; Wang, Zhihong; Koo, Youngmi; Zhao, Ansha; Sankar, Jagannathan; Kong, Deling; Huang, Nan; Yun, Yeoheung

2017-01-01

Current in vitro models fail in predicting the degradation rate and mode of magnesium (Mg) stents in vivo. To overcome this, the microenvironment of the stent is simulated here in an ex vivo bioreactor with porcine aorta and circulating medium, and compared with standard static in vitro immersion and with in vivo rat aorta models. In ex vivo and in vivo conditions, pure Mg wires were exposed to the aortic lumen and inserted into the aortic wall to mimic early- and long-term implantation, respectively. Results showed that: 1) Degradation rates of Mg were similar for all the fluid diffusion conditions (in vitro static, aortic wall ex vivo and in vivo); however, Mg degradation under flow condition (i.e. in the lumen) in vivo was slower than ex vivo; 2) The corrosion mode in the samples can be mainly described as localized (in vitro), mixed localized and uniform (ex vivo), and uniform (in vivo); 3) Abundant degradation products (MgO/Mg(OH)2 and Ca/P) with gas bubbles accumulated around the localized degradation regions ex vivo, but a uniform and thin degradation product layer was found in vivo. It is concluded that the ex vivo vascular bioreactor provides an improved test setting for magnesium degradation between static immersion and animal experiments and highlights its promising role in bridging degradation behavior and biological response for vascular stent research. PMID:28013101

Physiological and Molecular Effects of in vivo and ex vivo Mild Skin Barrier Disruption.

PubMed

Pfannes, Eva K B; Weiss, Lina; Hadam, Sabrina; Gonnet, Jessica; Combardière, Béhazine; Blume-Peytavi, Ulrike; Vogt, Annika

2018-01-01

The success of topically applied treatments on skin relies on the efficacy of skin penetration. In order to increase particle or product penetration, mild skin barrier disruption methods can be used. We previously described cyanoacrylate skin surface stripping as an efficient method to open hair follicles, enhance particle penetration, and activate Langerhans cells. We conducted ex vivo and in vivo measurements on human skin to characterize the biological effect and quantify barrier disruption-related inflammation on a molecular level. Despite the known immunostimulatory effects, this barrier disruption and hair follicle opening method was well accepted and did not result in lasting changes of skin physiological parameters, cytokine production, or clinical side effects. Only in ex vivo human skin did we find a discrete increase in IP-10, TGF-β, IL-8, and GM-CSF mRNA. The data underline the safety profile of this method and demonstrate that the procedure per se does not cause substantial inflammation or skin damage, which is also of interest when applied to non-invasive sampling of biomarkers in clinical trials. © 2018 S. Karger AG, Basel.

Imaging of prostate cancer: a platform for 3D co-registration of in-vivo MRI ex-vivo MRI and pathology

NASA Astrophysics Data System (ADS)

Orczyk, Clément; Mikheev, Artem; Rosenkrantz, Andrew; Melamed, Jonathan; Taneja, Samir S.; Rusinek, Henry

2012-02-01

Objectives: Multi-parametric MRI is emerging as a promising method for prostate cancer diagnosis. prognosis and treatment planning. However, the localization of in-vivo detected lesions and pathologic sites of cancer remains a significant challenge. To overcome this limitation we have developed and tested a system for co-registration of in-vivo MRI, ex-vivo MRI and histology. Materials and Methods: Three men diagnosed with localized prostate cancer (ages 54-72, PSA levels 5.1-7.7 ng/ml) were prospectively enrolled in this study. All patients underwent 3T multi-parametric MRI that included T2W, DCEMRI, and DWI prior to robotic-assisted prostatectomy. Ex-vivo multi-parametric MRI was performed on fresh prostate specimen. Excised prostates were then sliced at regular intervals and photographed both before and after fixation. Slices were perpendicular to the main axis of the posterior capsule, i.e., along the direction of the rectal wall. Guided by the location of the urethra, 2D digital images were assembled into 3D models. Cancer foci, extra-capsular extensions and zonal margins were delineated by the pathologist and included in 3D histology data. A locally-developed software was applied to register in-vivo, ex-vivo and histology using an over-determined set of anatomical landmarks placed in anterior fibro-muscular stroma, central. transition and peripheral zones. The mean root square distance across corresponding control points was used to assess co-registration error. Results: Two specimens were pT3a and one pT2b (negative margin) at pathology. The software successfully fused invivo MRI. ex-vivo MRI fresh specimen and histology using appropriate (rigid and affine) transformation models with mean square error of 1.59 mm. Coregistration accuracy was confirmed by multi-modality viewing using operator-guided variable transparency. Conclusion: The method enables successful co-registration of pre-operative MRI, ex-vivo MRI and pathology and it provides initial evidence

Ex vivo culture platform for assessment of cartilage repair treatment strategies.

PubMed

Schwab, Andrea; Meeuwsen, Annick; Ehlicke, Franziska; Hansmann, Jan; Mulder, Lars; Smits, Anthal; Walles, Heike; Kock, Linda

2017-01-01

There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors specific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, matrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivo testing.

Descemet Membrane Thickening as a Sign for the Diagnosis of Corneal Graft Rejection: An Ex Vivo Study.

PubMed

VanDenBerg, Ryan; Diakonis, Vasilios F; Bozung, Alison; Gameiro, Gustavo Rosa; Fischer, Oliver; El Dakkak, Ahmed; Ulloa-Padilla, Jan Paul; Anagnostopoulos, Apostolos; Dubovy, Sander; Abou Shousha, Mohamed

2017-12-01

To disclose, using an ex vivo study, the histopathological mechanism behind in vivo thickening of the endothelium/Descemet membrane complex (En/DM) observed in rejected corneal grafts (RCGs). Descemet membrane (DM), endothelium, and retrocorneal membranes make up the total En/DM thickness. These layers are not differentiable by high-definition optical coherence tomography; therefore, the source of thickening is unclear from an in vivo perspective. A retrospective ex vivo study (from September 2015 to December 2015) was conducted to measure the thicknesses of DM, endothelium, and retrocorneal membrane in 54 corneal specimens (31 RCGs and 23 controls) using light microscopy. Controls were globes with posterior melanoma without corneal involvement. There were 54 corneas examined ex vivo with mean age 58.1 ± 12.2 in controls and 51.7 ± 27.9 years in RCGs. The ex vivo study uncovered the histopathological mechanism of En/DM thickening to be secondary to significant thickening (P < 0.001) of DM (6.5 ± 2.4 μm) in RCGs compared with controls (3.9 ± 1.5 μm). Our ex vivo study shows that DM is responsible for thickening of the En/DM in RCGs observed in vivo by high-definition optical coherence tomography and not the endothelium or retrocorneal membrane.

Convection Enhanced Delivery: A Comparison of infusion characteristics in ex vivo and in vivo non-human primate brain tissue.

PubMed

Miranpuri, Gurwattan; Hinchman, Angelica; Wang, Anyi; Schomberg, Dominic; Kubota, Ken; Brady, Martin; Raghavan, Raghu; Bruner, Kevin; Brodsky, Ethan; Block, Walter; Grabow, Ben; Raschke, Jim; Alexander, Andrew; Ross, Chris; Simmons, Heather; Sillay, Karl

2013-07-01

Convection enhanced delivery (CED) is emerging as a promising infusion toolto facilitate delivery of therapeutic agents into the brain via mechanically controlled pumps. Infusion protocols and catheter design have an important impact on delivery. CED is a valid alternative for systemic administration of agents in clinical trials for cell and gene therapies. Where gel and ex vivo models are not sufficient in modeling the disease, in vivo models allow researchers to better understand the underlying mechanisms of neuron degeneration, which is helpful in finding novel approaches to control the process or reverse the progression. Determining the risks, benefits, and efficacy of new gene therapies introduced via CED will pave a way to enter human clinical trial. The objective of this study is to compare volume distribution (Vd)/ volume infused (Vi) ratios and backflow measurements following CED infusions in ex vivo versus in vivo non-human primate brain tissue, based on infusion protocols developed in vitro. In ex vivo infusions, the first brain received 2 infusions using a balloon catheter at rates of 1 μL/min and 2 μL/min for 30 minutes. The second and third brains received infusions using a valve-tip (VT) catheter at 1 μL/min for 30 minutes. The fourth brain received a total of 45 μL infused at a rate of 1 μL/min for 15 minutes followed by 2 μL/min for 15 minutes. Imaging was performed (SPGR FA34) every 3 minutes. In the in vivo group, 4 subjects received a total of 8 infusions of 50 μL. Subjects 1 and 2 received infusions at 1.0 μL/min using a VT catheter in the left hemisphere and a smart-flow (SF) catheter in the right hemisphere. Subjects 3 and 4 each received 1 infusion in the left and right hemisphere at 1.0 μL/min. MRI calculations of Vd/Vi did not significantly differ from those obtained on post-mortem pathology. The mean measured Vd/Vi of in vivo (5.23 + /-1.67) compared to ex vivo (2.17 + /-1.39) demonstrated a significantly larger Vd/Vi for in vivo

Sorbitol increases muscle glucose uptake ex vivo and inhibits intestinal glucose absorption ex vivo and in normal and type 2 diabetic rats.

PubMed

Chukwuma, Chika Ifeanyi; Islam, Md Shahidul

2017-04-01

Previous studies have suggested that sorbitol, a known polyol sweetener, possesses glycemic control potentials. However, the effect of sorbitol on intestinal glucose absorption and muscle glucose uptake still remains elusive. The present study investigated the effects of sorbitol on intestinal glucose absorption and muscle glucose uptake as possible anti-hyperglycemic or glycemic control potentials using ex vivo and in vivo experimental models. Sorbitol (2.5% to 20%) inhibited glucose absorption in isolated rat jejuna (IC 50 = 14.6% ± 4.6%) and increased glucose uptake in isolated rat psoas muscle with (GU 50 = 3.5% ± 1.6%) or without insulin (GU 50 = 7.0% ± 0.5%) in a concentration-dependent manner. Furthermore, sorbitol significantly delayed gastric emptying, accelerated digesta transit, inhibited intestinal glucose absorption, and reduced blood glucose increase in both normoglycemic and type 2 diabetic rats after 1 h of coingestion with glucose. Data of this study suggest that sorbitol exhibited anti-hyperglycemic potentials, possibly via increasing muscle glucose uptake ex vivo and reducing intestinal glucose absorption in normal and type 2 diabetic rats. Hence, sorbitol may be further investigated as a possible anti-hyperglycemic sweetener.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

PubMed Central

Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

2012-01-01

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

Arrhenius analysis of the relationship between hyperthermia and Hsp70 promoter activation: a comparison between ex vivo and in vivo data.

PubMed

Deckers, Roel; Debeissat, Christelle; Fortin, Pierre-Yves; Moonen, Chrit T W; Couillaud, Franck

2012-01-01

Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis. A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression. We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo. However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.

Equilibrium ex vivo calibration of homogenized tissue for in vivo SPME quantitation of doxorubicin in lung tissue.

PubMed

Roszkowska, Anna; Tascon, Marcos; Bojko, Barbara; Goryński, Krzysztof; Dos Santos, Pedro Reck; Cypel, Marcelo; Pawliszyn, Janusz

2018-06-01

The fast and sensitive determination of concentrations of anticancer drugs in specific organs can improve the efficacy of chemotherapy and minimize its adverse effects. In this paper, ex vivo solid-phase microextraction (SPME) coupled to LC-MS/MS as a method for rapidly quantitating doxorubicin (DOX) in lung tissue was optimized. Furthermore, the theoretical and practical challenges related to the real-time monitoring of DOX levels in the lung tissue of a living organism (in vivo SPME) are presented. In addition, several parameters for ex vivo/in vivo SPME studies, such as extraction efficiency of autoclaved fibers, intact/homogenized tissue differences, critical tissue amount, and the absence of an internal standard are thoroughly examined. To both accurately quantify DOX in solid tissue and minimize the error related to the lack of an internal standard, a calibration method at equilibrium conditions was chosen. In optimized ex vivo SPME conditions, the targeted compound was extracted by directly introducing a 15 mm (45 µm thickness) mixed-mode fiber into 15 g of homogenized tissue for 20 min, followed by a desorption step in an optimal solvent mixture. The detection limit for DOX was 2.5 µg g -1 of tissue. The optimized ex vivo SPME method was successfully applied for the analysis of DOX in real pig lung biopsies, providing an averaged accuracy and precision of 103.2% and 12.3%, respectively. Additionally, a comparison between SPME and solid-liquid extraction revealed good agreement. The results presented herein demonstrate that the developed SPME method radically simplifies the sample preparation step and eliminates the need for tissue biopsies. These results suggest that SPME can accurately quantify DOX in different tissue compartments and can be potentially useful for monitoring and adjusting drug dosages during chemotherapy in order to achieve effective and safe concentrations of doxorubicin. Copyright © 2018 Elsevier B.V. All rights reserved.

Accurate in vivo dielectric properties of liver from 500 MHz to 40 GHz and their correlation to ex vivo measurements.

PubMed

Farrugia, L; Wismayer, P Schembri; Mangion, L Zammit; Sammut, C V

2016-01-01

In this article, we report on the characterization of the dielectric properties of in vivo rat liver at 36.4°C from 500 MHz up to 40 GHz with less than 5% uncertainty. The measured data were fitted to a Cole-Cole model and dielectric parameters are presented together with their respective 95% confidence interval. The root mean square error is 0.42. Moreover, ex vivo measurements were conducted in situ at 1, 2, 4 and 6 min after animal death and are compared to in vivo measurements. The results show that immediate changes in [Formula: see text]and [Formula: see text] are within experimental uncertainty, and therefore changes between in vivo and published ex vivo dielectric properties can be attributed to tissue hydration.

Celiac Disease–Specific TG2-Targeted Autoantibodies Inhibit Angiogenesis Ex Vivo and In Vivo in Mice by Interfering with Endothelial Cell Dynamics

PubMed Central

Kalliokoski, Suvi; Sulic, Ana-Marija; Korponay-Szabó, Ilma R.; Szondy, Zsuzsa; Frias, Rafael; Perez, Mileidys Alea; Martucciello, Stefania; Roivainen, Anne; Pelliniemi, Lauri J.; Esposito, Carla; Griffin, Martin; Sblattero, Daniele; Mäki, Markku; Kaukinen, Katri; Lindfors, Katri; Caja, Sergio

2013-01-01

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility. PMID:23824706

Mucoadhesive Fenretinide Patches for Site-specific Chemoprevention of Oral Cancer: Enhancement of Oral Mucosal Permeation of Fenretinide by Co-incorporation of Propylene Glycol and Menthol

PubMed Central

Wu, Xiao; Desai, Kashappa-Goud H.; Mallery, Susan R.; Holpuch, Andrew S.; Phelps, Maynard P.; Schwendeman, Steven P.

2012-01-01

The objective of this study was to enhance oral mucosal permeation of fenretinide by co-incorporation of propylene glycol (PG) and menthol in fenretinide/Eudragit® RL PO mucoadhesive patches. Fenretinide is an extremely hydrophobic chemopreventive compound with poor tissue permeability. Co-incorporation of 5-10 wt% PG (mean Js = 16-23 μg cm−2 h−1; 158-171 μg fenretinide/g tissue) or 1-10 wt% PG + 5 wt% menthol (mean Js = 18-40 μg cm−2 h−1; 172-241 μg fenretinide/g tissue) in fenretinide/Eudragit® RL PO patches led to significant ex vivo fenretinide permeation enhancement (p < 0.001). Addition of PG above 2.5 wt% in the patch resulted in significant cellular swelling in the buccal mucosal tissues. These alterations were ameliorated by combining both enhancers and reducing PG level. After buccal administration of patches in rabbits, in vivo permeation of fenretinide across the oral mucosa was greater (~43 μg fenretinide/g tissue) from patches that contained optimized permeation enhancer content (2.5 wt% PG + 5 wt% menthol) relative to permeation obtained from enhancer-free patch (~ 17 μg fenretinide/g tissue) (p < 0.001). In vitro and in vivo release of fenretinide from patch was not significantly increased by co-incorporation of permeation enhancers, indicating that mass transfer across the tissue, and not the patch, largely determined the permeation rate control in vivo. As a result of its improved permeation and its lack of deleterious local effects, the mucoadhesive fenretinide patch co-incorporated with 2.5 wt% PG + 5 wt% menthol represents an important step in the further preclinical evaluation of oral site-specific chemoprevention strategies with fenretinide. PMID:22280430

Ultrasound-mediated nanoparticle delivery across ex vivo bovine retina after intravitreal injection.

PubMed

Huang, Di; Chen, Ying-Shan; Thakur, Sachin S; Rupenthal, Ilva D

2017-10-01

Intravitreal injection is the most common administration route for the treatment of retinal diseases. However, the vitreous and some of the retinal layers themselves act as significant barriers to efficient delivery of drugs administered intravitreally. This study aimed to improve the diffusive mobility of nanoparticles (NPs) in the vitreous and enhance their permeation across the retina after intravitreal injection by application of ultrasound (US). Ex vivo posterior bovine eye cups were used and the vitreous was either left intact or removed gently from the neural retina. Hyaluronic acid coated human serum albumin NPs were administered into the eye cups and continuous US with a frequency of 1MHz, an intensity of 0.5W/cm 2 , and a duration of 30s was applied once or repeatedly via the transscleral route. After pre-determined time points, fluorescence intensities in the vitreous and the retina were analyzed. Short pulses of US significantly improved the diffusive mobility of NPs through the vitreous as well as their penetration across the neural retina into the retinal pigment epithelium and choroid without causing any detectable damage to the ocular tissues. Therefore, transscleral US could be a powerful and safe tool to enhance retinal delivery of intravitreally injected NPs. Copyright © 2017 Elsevier B.V. All rights reserved.

21 CFR 582.6801 - Sodium tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium tartrate. 582.6801 Section 582.6801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Sodium tartrate. (a) Product. Sodium tartrate. (b) Conditions of use. This substance is generally...

21 CFR 582.6801 - Sodium tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium tartrate. 582.6801 Section 582.6801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Sodium tartrate. (a) Product. Sodium tartrate. (b) Conditions of use. This substance is generally...

21 CFR 582.6801 - Sodium tartrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium tartrate. 582.6801 Section 582.6801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Sodium tartrate. (a) Product. Sodium tartrate. (b) Conditions of use. This substance is generally...

21 CFR 582.6801 - Sodium tartrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium tartrate. 582.6801 Section 582.6801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Sodium tartrate. (a) Product. Sodium tartrate. (b) Conditions of use. This substance is generally...

21 CFR 582.6801 - Sodium tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium tartrate. 582.6801 Section 582.6801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Sodium tartrate. (a) Product. Sodium tartrate. (b) Conditions of use. This substance is generally...

Transdermal film-loaded finasteride microplates to enhance drug skin permeation: Two-step optimization study.

PubMed

Ahmed, Tarek A; El-Say, Khalid M

2016-06-10

The goal was to develop an optimized transdermal finasteride (FNS) film loaded with drug microplates (MIC), utilizing two-step optimization, to decrease the dosing schedule and inconsistency in gastrointestinal absorption. First; 3-level factorial design was implemented to prepare optimized FNS-MIC of minimum particle size. Second; Box-Behnken design matrix was used to develop optimized transdermal FNS-MIC film. Interaction among MIC components was studied using physicochemical characterization tools. Film components namely; hydroxypropyl methyl cellulose (X1), dimethyl sulfoxide (X2) and propylene glycol (X3) were optimized for their effects on the film thickness (Y1) and elongation percent (Y2), and for FNS steady state flux (Y3), permeability coefficient (Y4), and diffusion coefficient (Y5) following ex-vivo permeation through the rat skin. Morphological study of the optimized MIC and transdermal film was also investigated. Results revealed that stabilizer concentration and anti-solvent percent were significantly affecting MIC formulation. Optimized FNS-MIC of particle size 0.93μm was successfully prepared in which there was no interaction observed among their components. An enhancement in the aqueous solubility of FNS-MIC by more than 23% was achieved. All the studied variables, most of their interaction and quadratic effects were significantly affecting the studied variables (Y1-Y5). Morphological observation illustrated non-spherical, short rods, flakes like small plates that were homogeneously distributed in the optimized transdermal film. Ex-vivo study showed enhanced FNS permeation from film loaded MIC when compared to that contains pure drug. So, MIC is a successful technique to enhance aqueous solubility and skin permeation of poor water soluble drug especially when loaded into transdermal films. Copyright © 2016 Elsevier B.V. All rights reserved.

Ex vivo confocal microscopy: a new diagnostic technique for mucormycosis.

PubMed

Leclercq, A; Cinotti, E; Labeille, B; Perrot, J L; Cambazard, F

2016-05-01

Skin-dedicated ex vivo confocal microscopy (EVCM) has so far mainly been employed to identify cutaneous tumours on freshly excised samples. We present two cases where EVCM has been used to diagnose cutaneous mucormycosis. The skin biopsies were evaluated by the skin-dedicated ex vivo confocal microscope VivaScope 2500(®) (MAVIG GmbH, Munich Germany) under both reflectance and fluorescence mode. Conventional direct optical examination on skin scraping and histological examination were later performed. Mucormycetes observed by EVCM presented as hyper-reflective elongated 20 μm in diameter structures with perpendicular ramifications. Fungi were found both under reflectance and fluorescence mode and were better visible with acridine orange under fluorescence EVCM. Conventional direct optical examination on skin scraping and histological examination found the same elongated and branching structures confirming the presence of Mucormycetes. Ex vivo confocal microscopy has both the advantages of being fast as the direct optical examination, and to be able to show the localisation of the fungi in the tissue like the histological examination. In our cases, EVCM allowed to rapidly confirm the clinical diagnosis of mucormycosis, which is essential for the treatment of this fungal infection. Further studies are needed to compare the performance of EVCM with the findings of conventional histological and mycological examinations. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Graft downsizing during ex vivo lung perfusion: case report and technical notes.

PubMed

Nosotti, M; Rosso, L; Mendogni, P; Tosi, D; Palleschi, A; Righi, I; Froio, S; Valenza, F; Santambrogio, L

2014-09-01

Among patients with respiratory insufficiency awaiting lung transplantation, small adult patients have a lower opportunity of receiving size-matched pulmonary grafts, because of the shortage of donors, particularly those of small size. Reducing the size of an oversized graft is one of the methods to increase the donor pool; similarly, ex vivo lung perfusion is an emerging technique aimed toward the same purpose. We describe how we combined the 2 techniques (lobar transplantation plus contralateral nonanatomic graft reduction during ex vivo lung perfusion) to overcome graft shortage in a clinical case. For the 1st time, this case report demonstrates that surgical manipulation during ex vivo lung perfusion does not affect the functional improvement in a lung previously judged to be not suitable for transplantation. The 6-month follow-up results are similar to those of standard bilateral lung transplantation. Copyright © 2014 Elsevier Inc. All rights reserved.

Formulation, functional evaluation and ex vivo performance of thermoresponsive soluble gels - A platform for therapeutic delivery to mucosal sinus tissue.

PubMed

Pandey, Preeti; Cabot, Peter J; Wallwork, Benjamin; Panizza, Benedict J; Parekh, Harendra S

2017-01-01

Mucoadhesive in situ gelling systems (soluble gels) have received considerable attention recently as effective stimuli-transforming vectors for a range of drug delivery applications. Considering this fact, the present work involves systematic formulation development, optimization, functional evaluation and ex vivo performance of thermosensitive soluble gels containing dexamethasone 21-phosphate disodium salt (DXN) as the model therapeutic. A series of in situ gel-forming systems comprising the thermoreversible polymer poloxamer-407 (P407), along with hydroxypropyl methyl cellulose (HPMC) and chitosan were first formulated. The optimized soluble gels were evaluated for their potential to promote greater retention at the mucosal surface, for improved therapeutic efficacy, compared to existing solution/suspension-based steroid formulations used clinically. Optimized soluble gels demonstrated a desirable gelation temperature with Newtonian fluid behaviour observed under storage conditions (4-8°C), and pseudoplastic fluid behaviour recorded at nasal cavity/sinus temperature (≈34°C). The in vitro characterization of formulations including rheological evaluation, textural analysis and mucoadhesion studies of the gel form were investigated. Considerable improvement in mechanical properties and mucoadhesion was observed with incorporation of HPMC and chitosan into the gelling systems. The lead poloxamer-based soluble gels, PGHC4 and PGHC7, which were carried through to ex vivo permeation studies displayed extended drug release profiles in conditions mimicking the human nasal cavity, which indicates their suitability for treating a range of conditions affecting the nasal cavity/sinuses. Copyright © 2016 Elsevier B.V. All rights reserved.

Ex Vivo (Fluorescence) Confocal Microscopy in Surgical Pathology: State of the Art.

PubMed

Ragazzi, Moira; Longo, Caterina; Piana, Simonetta

2016-05-01

First developed in 1957, confocal microscopy is a powerful imaging tool that can be used to obtain near real-time reflected light images of untreated human tissue with nearly histologic resolution. Besides its research applications, in the last decades, confocal microscopy technology has been proposed as a useful device to improve clinical diagnosis, especially in ophthalmology, dermatology, and endomicroscopy settings, thanks to advances in instrument development. Compared with the wider use of the in vivo tissue assessment, ex vivo applications of confocal microscopy are not fully explored. A comprehensive review of the current literature was performed here, focusing on the reliable applications of ex vivo confocal microscopy in surgical pathology and on some potential evolutions of this new technique from pathologists' viewpoint.

Ex-vivo imaging of blood and lymphatic vessels in conjunctiva using optical coherence tomography

NASA Astrophysics Data System (ADS)

Gong, Peijun; Karnowski, Karol; Yu, Paula; An, Dong; Yu, Dao-Yi; Sampson, David D.

2017-04-01

Label-free imaging of the blood and lymphatic vessel networks of the conjunctiva of the eye is important in assessing the drainage pathways affected by glaucoma. We utilize the characteristically low signal in optical coherence tomography (OCT) provided by such vessels in ex vivo tissue to characterize their morphology in two and three dimensions. We demonstrate this method on conjunctiva from six porcine eyes, showing the ready visualization of both vessel networks. Such ex vivo characterization is a necessary precursor for future in vivo studies directed towards improving glaucoma surgery.

Bioadhesive films containing benzocaine: correlation between in vitro permeation and in vivo local anesthetic effect.

PubMed

de Araujo, Daniele Ribeiro; Padula, Cristina; Cereda, Cíntia Maria Saia; Tófoli, Giovana Radomille; Brito, Rui Barbosa; de Paula, Eneida; Nicoli, Sara; Santi, Patrizia

2010-08-01

The aim of this work was to develop anesthetic bioadhesive films containing benzocaine and study their in vitro skin permeation and in vivo performance, in comparison with commercial formulations. Films containing 3% and 5% w/w of benzocaine were prepared and characterized by weight, drug content, thickness and morphology. In vitro permeation assays were performed in vertical diffusion cells using full-thickness pig ear skin as barrier. Intensity and duration of analgesia were evaluated in rats by tail-flick test, and skin histological analysis was carried out. Tail-flick test showed that the duration of benzocaine-induced analgesia was significantly prolonged with the films compared to commercial creams, in agreement with the higher in vitro permeation. Histological analysis of the rat tail skin did not reveal morphological tissue changes nor cell infiltration signs after application of the commercial creams or films. Results from our study indicate that the films developed in this work can be considered as innovative dermal/transdermal therapeutic systems for benzocaine local delivery.

A dual-slot microwave antenna for more spherical ablation zones: ex vivo and in vivo validation.

PubMed

Chiang, Jason; Hynes, Kieran A; Bedoya, Mariajose; Brace, Christopher L

2013-08-01

To compare the performance of a microwave antenna design with two annular slots to that of a monopole antenna design in creating a more spherical ablation zone. Animal care and use committee approval was obtained before in vivo experiments were performed. Microwave ablation zones were created by using dual-slot and monopole control antennas for 2, 5, and 10 minutes at 50 and 100 W in ex vivo bovine livers. Dual-slot and monopole antennas were then used to create ablation zones at 100 W for 5 minutes in in vivo porcine livers, which also underwent intraprocedural imaging. Ablation diameter, length, and aspect ratio (diameter ÷ length) were measured at gross pathologic examination and compared at each combination of power and time by using the paired Student t test. A P value less than .05 was considered to indicate a significant difference. Aspect ratios closer to 1 reflected a more spherical ablation zone. The dual-slot antenna created ablation zones with a higher aspect ratio at 50 W for 2 minutes (0.75 vs 0.53, P = .003) and 5 minutes (0.82 vs 0.63, P = .053) than did the monopole antenna in ex vivo liver tissue, although the difference was only significant at 2 minutes. At 100 W, the dual-slot antenna had a significantly higher aspect ratio at 2 minutes (0.52 vs 0.42, P = .002). In vivo studies showed significantly higher aspect ratios at 100 W for 5 minutes (0.63 vs 0.53, respectively, P = .029). Intraprocedural imaging confirmed this characterization, showing higher rates of ablation zone growth and heating primarily at the early stages of the ablation procedure when the dual-slot antenna was used. The dual-slot microwave antenna created a more spherical ablation zone than did the monopole antenna both in vivo and ex vivo liver tissue. Greater control over power delivery can potentially extend the advantages of the dual-slot antenna design to higher power and longer treatment times.

A new veno-venous bypass type for ex-vivo liver resection in dogs.

PubMed

Lei, Peng; Liu, Shi-Qi; Cui, Xiao-Hai; Lv, Yi; Zhao, Ge; Li, Jian-Hui

2013-08-01

Ex-vivo liver resection is a procedure in which the liver is completely removed, perfused and after bench surgery, the liver is autotransplanted to the original site. Ex-vivo liver resection is an important treatment for unresectable liver tumors. This surgical procedure requires long operation time, during which blood flow must be carefully maintained to avoid venous congestion. An effective veno-venous bypass (VVB) may meet this requirement. The present study was to test our new designed VVB device which comprised one heparinized polyvinylchloride tube and three magnetic rings. The efficacy of this device was tested in five dogs. A VVB was established in 6-10 minutes. There was no leakage during the procedure. Hemodynamics was stable at anhepatic phase, which indicated that the bypass was successful. This newly-developed VVB device maintained circulation stability during ex-vivo liver resection in our dog model and thus, this VVB device significantly shortened the operation time.

The use of rats and mice as animal models in ex vivo bone growth and development studies

PubMed Central

Abubakar, A. A.; Noordin, M. M.; Azmi, T. I.; Kaka, U.

2016-01-01

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine. Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2. PMID:27965220

Solubility and transdermal permeation properties of a dehydroepiandrosterone cyclodextrin complex from hydrophilic and lipophilic vehicles.

PubMed

Ceschel, GianCarlo; Bergamante, Valentina; Maffei, Paola; Lombardi Borgia, Simone; Calabrese, Valeria; Biserni, Stefano; Ronchi, Celestino

2005-01-01

The permeation ability of a compound is due principally to its concentration in the vehicle and to its aptitude to cross the stratum corneum of the skin. In this work ex-vivo permeation studies on newly developed formulations containing dehydroepiandrosterone (DHEA) were carried out to investigate vehicles that increase drug permeation through the skin. To enhance the solubility of DHEA, its complex form with alpha-cyclodextrin was used. In addition, the two forms (pure drug and complex form) were introduced in hydrophilic (water), lipophilic (paraffin oil), and microemulsion vehicles to evaluate the synergic effect of cyclodextrins and microemulsion vehicles on solubility and permeation. From the results, DHEA solubility is notably conditioned by the type of the vehicle used: the highest solubilities (both for pure and complex drug forms) were obtained with microemulsion, followed by paraffin oil and water. Moreover, in all the studied vehicles, the c-DHEA was more soluble than DHEA. Permeation profile fluxes showed very interesting differences. That reflect the varying drug forms (pure drug and complex form), vehicles used, and drug concentrations in the vehicles. The major flux was obtained in complex of DHEA with alpha-cyclodextrins in the microemulsion vehicle. Therefore, this type of vehicle and drug form would be very useful in the development of a topical formulation containing DHEA.

Large-Animal Biventricular Working Heart Perfusion System with Low Priming Volume-Comparison between in vivo and ex vivo Cardiac Function.

PubMed

Abicht, Jan-Michael; Mayr, Tanja Axinja Jelena; Jauch, Judith; Guethoff, Sonja; Buchholz, Stefan; Reichart, Bruno; Bauer, Andreas

2018-01-01

Existing large-animal, ex vivo, cardiac perfusion models are restricted in their ability to establish an ischemia/reperfusion condition as seen in cardiac surgery or transplantation. Other working heart systems only challenge one ventricle or require a substantially larger priming volume. We describe a novel biventricular cardiac perfusion system with reduced priming volume. Juvenile pig hearts were cardiopleged, explanted, and reperfused ex vivo after 150 minutes of cold ischemia. Autologous whole blood was used as perfusate (minimal priming volume 350 mL). After 15 minutes of Langendorff perfusion (LM), the system was switched into a biventricular working mode (WM) and studied for 3 hours. During reperfusion, complete unloading of both ventricles and constant-pressure coronary perfusion was achieved. During working mode perfusion, the preload and afterload pressure of both ventricles was controlled within the targeted physiologic range. Functional parameters such as left ventricular work index were reduced in ex vivo working mode (in vivo: 787 ± 186 vs. 1 h WM 498 ± 66 mm Hg·mL/g·min; p  < 0.01), but remained stable throughout the following study period (3 h WM 517 ± 103 mm Hg·mL/g·min; p  = 0.63). Along with the elevated workload during WM, myocardial metabolism and oxygen consumption increased compared with LM (0.021 ± 0.08 vs. 0.06 ± 0.01 mL/min/g; 1 h after reperfusion). Histologic examination of the myocardium revealed no structural damage. In the ex vivo perfusion system, stable hemodynamic and metabolic conditions can be established for a period of 3 hours while functional and blood parameters are easily accessible. Moreover, because of the minimal priming volume, the novel ex vivo cardiac perfusion circuit allows for autologous perfusion, using the limited amount of blood available from the organ donating animal. Georg Thieme Verlag KG Stuttgart · New York.

Optimized magnetic resonance diffusion protocol for ex-vivo whole human brain imaging with a clinical scanner

NASA Astrophysics Data System (ADS)

Scherrer, Benoit; Afacan, Onur; Stamm, Aymeric; Singh, Jolene; Warfield, Simon K.

2015-03-01

Diffusion-weighted magnetic resonance imaging (DW-MRI) provides a novel insight into the brain to facilitate our understanding of the brain connectivity and microstructure. While in-vivo DW-MRI enables imaging of living patients and longitudinal studies of brain changes, post-mortem ex-vivo DW-MRI has numerous advantages. Ex-vivo imaging benefits from greater resolution and sensitivity due to the lack of imaging time constraints; the use of tighter fitting coils; and the lack of movement artifacts. This allows characterization of normal and abnormal tissues with unprecedented resolution and sensitivity, facilitating our ability to investigate anatomical structures that are inaccessible in-vivo. This also offers the opportunity to develop today novel imaging biomarkers that will, with tomorrow's MR technology, enable improved in-vivo assessment of the risk of disease in an individual. Post-mortem studies, however, generally rely on the fixation of specimen to inhibit tissue decay which starts as soon as tissue is deprived from its blood supply. Unfortunately, fixation of tissues substantially alters tissue diffusivity profiles. In addition, ex-vivo DW-MRI requires particular care when packaging the specimen because the presence of microscopic air bubbles gives rise to geometric and intensity image distortion. In this work, we considered the specific requirements of post-mortem imaging and designed an optimized protocol for ex-vivo whole brain DW-MRI using a human clinical 3T scanner. Human clinical 3T scanners are available to a large number of researchers and, unlike most animal scanners, have a bore diameter large enough to image a whole human brain. Our optimized protocol will facilitate widespread ex-vivo investigations of large specimen.

Carlecortemcel-l: an ex vivo expanded umbilical cord blood cell graft for allogeneic transplantation.

PubMed

Petropoulos, Demetrios; Chan, Ka Wah

2009-11-01

Success of umbilical cord blood transplantation (UCBT) is mostly affected by the cell dose infused and its application is limited by the size of the recipient. For most adults and older children it is not possible to find a single UCB unit large enough for reliable engraftment. One strategy to increase the number of progenitor cells available is ex vivo expansion of the unit. The main challenge of ex vivo expansion systems is how not to deplete the self-renewing cell population by driving them into differentiation into committed progenitors. Copper modulates basic cell functions, such as survival, proliferation, and differentiation. Reduction of cellular copper in ex vivo culture conditions enabled preferential proliferation of early progenitors and increased engraftment capabilities. The result of a Phase I study of carlecortemcel-l, a product derived from ex vivo expansion of UCB progenitors in the presence of a copper chelator and early-acting cytokines, and the study design for the current pivotal study are presented. A literature review using PubMed and the investigator's brochure from the manufacturer. Early results suggest that carlecortemcel-l infusion is safe and may be associated with favorable non-relapse mortality rates. A pivotal global study is currently being conducted to evaluate safety and efficacy of this product from centralized manufacturing facilities.

21 CFR 184.1801 - Sodium tartrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium tartrate. 184.1801 Section 184.1801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Specific Substances Affirmed as GRAS § 184.1801 Sodium tartrate. (a) Sodium tartrate (C4H4Na2O6·2H2O, CAS...

21 CFR 184.1801 - Sodium tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium tartrate. 184.1801 Section 184.1801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Specific Substances Affirmed as GRAS § 184.1801 Sodium tartrate. (a) Sodium tartrate (C4H4Na2O6·2H2O, CAS...

21 CFR 184.1801 - Sodium tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium tartrate. 184.1801 Section 184.1801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Specific Substances Affirmed as GRAS § 184.1801 Sodium tartrate. (a) Sodium tartrate (C4H4Na2O6·2H2O, CAS...

Vaginal Lactobacillus Inhibits HIV-1 Replication in Human Tissues Ex Vivo

PubMed Central

Ñahui Palomino, Rogers A.; Zicari, Sonia; Vanpouille, Christophe; Vitali, Beatrice; Margolis, Leonid

2017-01-01

Lactobacillus species, which dominate vaginal microbiota of healthy reproductive-age women, lower the risks of sexually transmitted infections, including the risk of human immunodeficiency virus (HIV) acquisition. The exact mechanisms of this protection remain to be understood. Here, we investigated these mechanisms in the context of human cervico-vaginal and lymphoid tissues ex vivo. We found that all six Lactobacillus strains tested in these systems significantly suppressed HIV type-1 (HIV-1) infection. We identified at least three factors that mediated this suppression: (i) Acidification of the medium. The pH of the undiluted medium conditioned by lactobacilli was between 3.8 and 4.6. Acidification of the culture medium with hydrochloric acid (HCl) to this pH in control experiments was sufficient to abrogate HIV-1 replication. However, the pH of the Lactobacillus-conditioned medium (CM) diluted fivefold, which reached ∼6.9, was also suppressive for HIV-1 infection, while in control experiments HIV-1 infection was not abrogated when the pH of the medium was brought to 6.9 through the use of HCl. This suggested the existence of other factors responsible for HIV-1 inhibition by lactobacilli. (ii) Lactic acid. There was a correlation between the concentration of lactic acid in the Lactobacillus-CM and its ability to suppress HIV-1 infection in human tissues ex vivo. Addition of lactic acid isomers D and L to tissue culture medium at the concentration that corresponded to their amount released by lactobacilli resulted in HIV-1 inhibition. Isomer L was produced in higher quantities than isomer D and was mostly responsible for HIV-1 inhibition. These results indicate that lactic acid, in particular its L-isomer, inhibits HIV-1 independently of lowering of the pH. (iii) Virucidal effect. Incubation of HIV-1 in Lactobacillus-CM significantly suppressed viral infectivity for human tissues ex vivo. Finally, lactobacilli adsorb HIV-1, serving as a sink decreasing the

Development of sustained release capsules containing "coated matrix granules of metoprolol tartrate".

PubMed

Siddique, Sabahuddin; Khanam, Jasmina; Bigoniya, Papiya

2010-09-01

The objective of this investigation was to prepare sustained release capsule containing coated matrix granules of metoprolol tartrate and to study its in vitro release and in vivo absorption. The design of dosage form was performed by choosing hydrophilic hydroxypropyl methyl cellulose (HPMC K100M) and hydrophobic ethyl cellulose (EC) polymers as matrix builders and Eudragit® RL/RS as coating polymers. Granules were prepared by composing drug with HPMC K100M, EC, dicalcium phosphate by wet granulation method with subsequent coating. Optimized formulation of metoprolol tartrate was formed by using 30% HPMC K100M, 20% EC, and ratio of Eudragit® RS/RL as 97.5:2.5 at 25% coating level. Capsules were filled with free flowing optimized granules of uniform drug content. This extended the release period upto 12 h in vitro study. Similarity factor and mean dissolution time were also reported to compare various dissolution profiles. The network formed by HPMC and EC had been coupled satisfactorily with the controlled resistance offered by Eudragit® RS. The release mechanism of capsules followed Korsemeyer-Peppas model that indicated significant contribution of erosion effect of hydrophilic polymer. Biopharmaceutical study of this optimized dosage form in rabbit model showed 10 h prolonged drug release in vivo. A close correlation (R(2) = 0.9434) was established between the in vitro release and the in vivo absorption of drug. The results suggested that wet granulation with subsequent coating by fluidized bed technique, is a suitable method to formulate sustained release capsules of metoprolol tartrate and it can perform therapeutically better than conventional immediate release dosage form.

Measuring in-vivo and in-situ ex-vivo the 3D deformation of the lamina cribrosa microstructure under elevated intraocular pressure

NASA Astrophysics Data System (ADS)

Wei, Junchao; Yang, Bin; Voorhees, Andrew P.; Tran, Huong; Brazile, Bryn; Wang, Bo; Schuman, Joel; Smith, Matthew A.; Wollstein, Gadi; Sigal, Ian A.

2018-02-01

Elevated intraocular pressure (IOP) deforms the lamina cribrosa (LC), a structure within the optic nerve head (ONH) in the back of the eye. Evidence suggests that these deformations trigger events that eventually cause irreversible blindness, and have therefore been studied in-vivo using optical coherence tomography (OCT), and ex-vivo using OCT and a diversity of techniques. To the best of our knowledge, there have been no in-situ ex-vivo studies of LC mechanics. Our goal was two-fold: to introduce a technique for measuring 3D LC deformations from OCT, and to determine whether deformations of the LC induced by elevated IOP differ between in-vivo and in-situ ex-vivo conditions. A healthy adult rhesus macaque monkey was anesthetized and IOP was controlled by inserting a 27- gauge needle into the anterior chamber of the eye. Spectral domain OCT was used to obtain volumetric scans of the ONH at normal and elevated IOPs. To improve the visibility of the LC microstructure the scans were first processed using a novel denoising technique. Zero-normalized cross-correlation was used to find paired corresponding locations between images. For each location pair, the components of the 3D strain tensor were determined using non-rigid image registration. A mild IOP elevation from 10 to 15mmHg caused LC effective strains as large as 3%, and about 50% larger in-vivo than in-situ ex-vivo. The deformations were highly heterogeneous, with substantial 3D components, suggesting that accurate measurement of LC microstructure deformation requires high-resolution volumes. This technique will help improve understanding of LC biomechanics and how IOP contributes to glaucoma.

The correlation of in vivo and ex vivo tissue dielectric properties to validate electromagnetic breast imaging: initial clinical experience

PubMed Central

Halter, Ryan J; Zhou, Tian; Meaney, Paul M; Hartov, Alex; Barth, Richard J; Rosenkranz, Kari M; Wells, Wendy A; Kogel, Christine A; Borsic, Andrea; Rizzo, Elizabeth J; Paulsen, Keith D

2009-01-01

Electromagnetic (EM) breast imaging provides low-cost, safe and potentially a more specific modality for cancer detection than conventional imaging systems. A primary difficulty in validating these EM imaging modalities is that the true dielectric property values of the particular breast being imaged are not readily available on an individual subject basis. Here, we describe our initial experience in seeking to correlate tomographic EM imaging studies with discrete point spectroscopy measurements of the dielectric properties of breast tissue. The protocol we have developed involves measurement of in vivo tissue properties during partial and full mastectomy procedures in the operating room (OR) followed by ex vivo tissue property recordings in the same locations in the excised tissue specimens in the pathology laboratory immediately after resection. We have successfully applied all of the elements of this validation protocol in a series of six women with cancer diagnoses. Conductivity and permittivity gauged from ex vivo samples over the frequency range 100 Hz–8.5 GHz are found to be similar to those reported in the literature. A decrease in both conductivity and permittivity is observed when these properties are gauged from ex vivo samples instead of in vivo. We present these results in addition to a case study demonstrating how discrete point spectroscopy measurements of the tissue can be correlated and used to validate EM imaging studies. PMID:19491436

DNA damage in lens epithelium of cataract patients in vivo and ex vivo.

PubMed

Øsnes-Ringen, Oyvind; Azqueta, Amaia O; Moe, Morten C; Zetterström, Charlotta; Røger, Magnus; Nicolaissen, Bjørn; Collins, Andrew R

2013-11-01

DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro.

PubMed

Laihia, J K; Jansen, C T

2000-08-01

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.

21 CFR 184.1801 - Sodium tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium tartrate. 184.1801 Section 184.1801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT... GRAS § 184.1801 Sodium tartrate. (a) Sodium tartrate (C4H4Na2O6·2H2O, CAS Reg. No. 868-18-8) is the...

21 CFR 184.1801 - Sodium tartrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium tartrate. 184.1801 Section 184.1801 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1801 Sodium tartrate. (a) Sodium tartrate (C4H4Na2O6·2H2O, CAS Reg. No. 868...

Combined in vivo and ex vivo analysis of mesh mechanics in a porcine hernia model.

PubMed

Kahan, Lindsey G; Lake, Spencer P; McAllister, Jared M; Tan, Wen Hui; Yu, Jennifer; Thompson, Dominic; Brunt, L Michael; Blatnik, Jeffrey A

2018-02-01

Hernia meshes exhibit variability in mechanical properties, and their mechanical match to tissue has not been comprehensively studied. We used an innovative imaging model of in vivo strain tracking and ex vivo mechanical analysis to assess effects of mesh properties on repaired abdominal walls in a porcine model. We hypothesized that meshes with dissimilar mechanical properties compared to native tissue would alter abdominal wall mechanics more than better-matched meshes. Seven mini-pigs underwent ventral hernia creation and subsequent open repair with one of two heavyweight polypropylene meshes. Following mesh implantation with attached radio-opaque beads, fluoroscopic images were taken at insufflation pressures from 5 to 30 mmHg on postoperative days 0, 7, and 28. At 28 days, animals were euthanized and ex vivo mechanical testing performed on full-thickness samples across repaired abdominal walls. Testing was conducted on 13 mini-pig controls, and on meshes separately. Stiffness and anisotropy (the ratio of stiffness in the transverse versus craniocaudal directions) were assessed. 3D reconstructions of repaired abdominal walls showed stretch patterns. As pressure increased, both meshes expanded, with no differences between groups. Over time, meshes contracted 17.65% (Mesh A) and 0.12% (Mesh B; p = 0.06). Mesh mechanics showed that Mesh A deviated from anisotropic native tissue more than Mesh B. Compared to native tissue, Mesh A was stiffer both transversely and craniocaudally. Explanted repaired abdominal walls of both treatment groups were stiffer than native tissue. Repaired tissue became less anisotropic over time, as mesh properties prevailed over native abdominal wall properties. This technique assessed 3D stretch at the mesh level in vivo in a porcine model. While the abdominal wall expanded, mesh-ingrown areas contracted, potentially indicating stresses at mesh edges. Ex vivo mechanics demonstrate that repaired tissue adopts mesh properties, suggesting

Design, characterization and ex vivo evaluation of chitosan film integrating of insulin nanoparticles composed of thiolated chitosan derivative for buccal delivery of insulin.

PubMed

Mortazavian, Elaheh; Dorkoosh, Farid Abedin; Rafiee-Tehrani, Morteza

2014-05-01

The purpose of this study is to optimize and characterize of chitosan buccal film for delivery of insulin nanoparticles that were prepared from thiolated dimethyl ethyl chitosan (DMEC-Cys). Insulin nanoparticles composed of chitosan and dimethyl ethyl chitosan (DMEC) were also prepared as control groups. The release of insulin from nanoparticles was studied in vitro in phosphate buffer solution (PBS) pH 7.4. Optimization of chitosan buccal films has been carried out by central composite design (CCD) response surface methodology. Independent variables were different amounts of chitosan and glycerol as mucoadhesive polymer and plasticizer, respectively. Tensile strength and bioadhesion force were considered as dependent variables. Ex vivo study was performed on excised rabbit buccal mucosa. Optimized insulin nanoparticles were obtained with acceptable physicochemical properties. In vitro release profile of insulin nanoparticles revealed that the highest solubility of nanoparticles in aqueous media is related to DMEC-Cys nanoparticles. CCD showed that optimized buccal film containing 4% chitosan and 10% glycerol has 5.81 kg/mm(2) tensile strength and 2.47 N bioadhesion forces. Results of ex vivo study demonstrated that permeation of insulin nanoparticles through rabbit buccal mucosa is 17.1, 67.89 and 97.18% for chitosan, DMEC and DMEC-Cys nanoparticles, respectively. Thus, this study suggests that DMEC-Cys can act as a potential enhancer for buccal delivery of insulin.

Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo1

PubMed Central

Shedlock, Devon J.; Talbott, Kendra T.; Morrow, Matthew P.; Ferraro, Bernadette; Hokey, David A.; Muthumani, Karuppiah; Weiner, David B.

2010-01-01

The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly-used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here we describe an alternative, semi-quantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for five days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays. PMID:20104580

Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells

PubMed Central

Fu, Xing; Du, Min

2018-01-01

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health. PMID:29643147

Ex vivo blood vessel bioreactor for analysis of the biodegradation of magnesium stent models with and without vessel wall integration.

PubMed

Wang, Juan; Liu, Lumei; Wu, Yifan; Maitz, Manfred F; Wang, Zhihong; Koo, Youngmi; Zhao, Ansha; Sankar, Jagannathan; Kong, Deling; Huang, Nan; Yun, Yeoheung

2017-03-01

Current in vitro models fail in predicting the degradation rate and mode of magnesium (Mg) stents in vivo. To overcome this, the microenvironment of the stent is simulated here in an ex vivo bioreactor with porcine aorta and circulating medium, and compared with standard static in vitro immersion and with in vivo rat aorta models. In ex vivo and in vivo conditions, pure Mg wires were exposed to the aortic lumen and inserted into the aortic wall to mimic early- and long-term implantation, respectively. Results showed that: 1) Degradation rates of Mg were similar for all the fluid diffusion conditions (in vitro static, aortic wall ex vivo and in vivo); however, Mg degradation under flow condition (i.e. in the lumen) in vivo was slower than ex vivo; 2) The corrosion mode in the samples can be mainly described as localized (in vitro), mixed localized and uniform (ex vivo), and uniform (in vivo); 3) Abundant degradation products (MgO/Mg(OH) 2 and Ca/P) with gas bubbles accumulated around the localized degradation regions ex vivo, but a uniform and thin degradation product layer was found in vivo. It is concluded that the ex vivo vascular bioreactor provides an improved test setting for magnesium degradation between static immersion and animal experiments and highlights its promising role in bridging degradation behavior and biological response for vascular stent research. Magnesium and its alloys are candidates for a new generation of biodegradable stent materials. However, the in vitro degradation of magnesium stents does not match the clinical degradation rates, corrupting the validity of conventional degradation tests. Here we report an ex vivo vascular bioreactor, which allows simulation of the microenvironment with and without blood vessel integration to study the biodegradation of magnesium implants in comparison with standard in vitro test conditions and with in vivo implantations. The bioreactor did simulate the corrosion of an intramural implant very well, but

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.

PubMed

Jenkins, Russell W; Aref, Amir R; Lizotte, Patrick H; Ivanova, Elena; Stinson, Susanna; Zhou, Chensheng W; Bowden, Michaela; Deng, Jiehui; Liu, Hongye; Miao, Diana; He, Meng Xiao; Walker, William; Zhang, Gao; Tian, Tian; Cheng, Chaoran; Wei, Zhi; Palakurthi, Sangeetha; Bittinger, Mark; Vitzthum, Hans; Kim, Jong Wook; Merlino, Ashley; Quinn, Max; Venkataramani, Chandrasekar; Kaplan, Joshua A; Portell, Andrew; Gokhale, Prafulla C; Phillips, Bart; Smart, Alicia; Rotem, Asaf; Jones, Robert E; Keogh, Lauren; Anguiano, Maria; Stapleton, Lance; Jia, Zhiheng; Barzily-Rokni, Michal; Cañadas, Israel; Thai, Tran C; Hammond, Marc R; Vlahos, Raven; Wang, Eric S; Zhang, Hua; Li, Shuai; Hanna, Glenn J; Huang, Wei; Hoang, Mai P; Piris, Adriano; Eliane, Jean-Pierre; Stemmer-Rachamimov, Anat O; Cameron, Lisa; Su, Mei-Ju; Shah, Parin; Izar, Benjamin; Thakuria, Manisha; LeBoeuf, Nicole R; Rabinowits, Guilherme; Gunda, Viswanath; Parangi, Sareh; Cleary, James M; Miller, Brian C; Kitajima, Shunsuke; Thummalapalli, Rohit; Miao, Benchun; Barbie, Thanh U; Sivathanu, Vivek; Wong, Joshua; Richards, William G; Bueno, Raphael; Yoon, Charles H; Miret, Juan; Herlyn, Meenhard; Garraway, Levi A; Van Allen, Eliezer M; Freeman, Gordon J; Kirschmeier, Paul T; Lorch, Jochen H; Ott, Patrick A; Hodi, F Stephen; Flaherty, Keith T; Kamm, Roger D; Boland, Genevieve M; Wong, Kwok-Kin; Dornan, David; Paweletz, Cloud Peter; Barbie, David A

2018-02-01

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR. See related commentary by Balko and Sosman, p. 143 See related article by Deng et al., p. 216 This article is highlighted in the In This Issue feature, p. 127 . ©2017 American Association for Cancer Research.

21 CFR 582.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium potassium tartrate. 582.1804 Section 582.1804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Additives § 582.1804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of...

21 CFR 582.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium potassium tartrate. 582.1804 Section 582.1804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Additives § 582.1804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of...

21 CFR 582.6804 - Sodium potassium tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium potassium tartrate. 582.6804 Section 582.6804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....6804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of use. This...

21 CFR 582.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium potassium tartrate. 582.1804 Section 582.1804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Additives § 582.1804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of...

21 CFR 582.6804 - Sodium potassium tartrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium potassium tartrate. 582.6804 Section 582.6804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....6804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of use. This...

21 CFR 582.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium potassium tartrate. 582.1804 Section 582.1804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Additives § 582.1804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of...

21 CFR 582.6804 - Sodium potassium tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium potassium tartrate. 582.6804 Section 582.6804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....6804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of use. This...

21 CFR 582.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium potassium tartrate. 582.1804 Section 582.1804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Additives § 582.1804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of...

21 CFR 582.6804 - Sodium potassium tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium potassium tartrate. 582.6804 Section 582.6804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....6804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of use. This...

21 CFR 582.6804 - Sodium potassium tartrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium potassium tartrate. 582.6804 Section 582.6804 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....6804 Sodium potassium tartrate. (a) Product. Sodium potassium tartrate. (b) Conditions of use. This...

Zolpidem tartrate and somnambulism.

PubMed

Harazin, J; Berigan, T R

1999-09-01

A case is reported in which a patient experienced somnambulistic episodes only after taking zolpidem tartrate for insomnia. Previous to the patient's use of zolpidem tartrate he had never experienced sleepwalking, and once the medication was discontinued the sleepwalking stopped. A search of the literature revealed only two other cases of zolpidem-induced sleepwalking, both involving individuals with a previous history of somnambulism in their youth.

21 CFR 582.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Potassium acid tartrate. 582.1077 Section 582.1077 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1077 Potassium acid tartrate. (a) Product. Potassium acid tartrate. (b) Conditions of use...

21 CFR 582.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Potassium acid tartrate. 582.1077 Section 582.1077 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1077 Potassium acid tartrate. (a) Product. Potassium acid tartrate. (b) Conditions of use...

21 CFR 582.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Potassium acid tartrate. 582.1077 Section 582.1077 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1077 Potassium acid tartrate. (a) Product. Potassium acid tartrate. (b) Conditions of use...

21 CFR 582.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Potassium acid tartrate. 582.1077 Section 582.1077 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1077 Potassium acid tartrate. (a) Product. Potassium acid tartrate. (b) Conditions of use...

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Code of Federal Regulations, 2010 CFR

2010-04-01

... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo Cultivation

PubMed Central

Granzin, Markus; Wagner, Juliane; Köhl, Ulrike; Cerwenka, Adelheid; Huppert, Volker; Ullrich, Evelyn

2017-01-01

Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context, ex vivo cultivation is an attractive option to increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore, ex vivo cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario, ex vivo cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different ex vivo cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant

Analysis of antigen-specific B-cell memory directly ex vivo.

PubMed

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Training gastroenterology fellows to perform gastric polypectomy using a novel ex vivo model

PubMed Central

Chen, Ming-Jen; Lin, Ching-Chung; Liu, Chia-Yuan; Chen, Chih-Jen; Chang, Chen-Wang; Chang, Ching-Wei; Lee, Chien-Wei; Shih, Shou-Chuan; Wang, Horng-Yuan

2011-01-01

AIM: To evaluate the effect of hands-on training of gastroenterology fellows in gastric polypectomy using an ex vivo simulator. METHODS: Eight gastroenterology fellows at Mackay Memorial Hospital, Taipei were evaluated in gastric polypectomy techniques using a pig stomach with artificial polyps created by a rubber band ligation device. The performance of four second year (year-2) fellows who had undergone one year of clinical training was compared with that of four first year (year-1) fellows both before and after a 4-h workshop using the ex vivo simulator. The workshop allowed for hands-on training in the removal of multiple artificial polyps and the placement of hemoclips at the excision site. Evaluation included observation of technical skills, procedure time, and the fellows’ confidence scale. RESULTS: One week after the workshop, the year-1 fellows were re-evaluated and had significantly improved mean performance scores (from 17.9 ± 1.8 to 22.5 ± 0.7), confidence scale (from 4.5 ± 1.0 to 7.8 ± 0.5) and procedure time (from 615.0 ± 57.4 s to 357.5 ± 85.0 s) compared with their baseline performance. After 4 h of training using the ex vivo simulator, the skills of the year-1 fellows were statistically similar to those of the year-2 fellows. CONCLUSION: Use of this ex vivo simulator significantly improved the endoscopic gastric polypectomy skills of gastroenterology fellows who had not had previous clinical training in gastric polypectomy. PMID:22147969

Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo.

PubMed

Nedosekin, Dmitry A; Juratli, Mazen A; Sarimollaoglu, Mustafa; Moore, Christopher L; Rusch, Nancy J; Smeltzer, Mark S; Zharov, Vladimir P; Galanzha, Ekaterina I

2013-06-01

Circulating cells, bacteria, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dielectric properties of human normal, malignant and cirrhotic liver tissue: in vivo and ex vivo measurements from 0.5 to 20 GHz using a precision open-ended coaxial probe.

PubMed

O'Rourke, Ann P; Lazebnik, Mariya; Bertram, John M; Converse, Mark C; Hagness, Susan C; Webster, John G; Mahvi, David M

2007-08-07

Hepatic malignancies have historically been treated with surgical resection. Due to the shortcomings of this technique, there is interest in other, less invasive, treatment modalities, such as microwave hepatic ablation. Crucial to the development of this technique is the accurate knowledge of the dielectric properties of human liver tissue at microwave frequencies. To this end, we characterized the dielectric properties of in vivo and ex vivo normal, malignant and cirrhotic human liver tissues from 0.5 to 20 GHz. Analysis of our data at 915 MHz and 2.45 GHz indicates that the dielectric properties of ex vivo malignant liver tissue are 19 to 30% higher than normal tissue. The differences in the dielectric properties of in vivo malignant and normal liver tissue are not statistically significant (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 16% higher than normal). Also, the dielectric properties of in vivo normal liver tissue at 915 MHz and 2.45 GHz are 16 to 43% higher than ex vivo. No statistically significant differences were found between the dielectric properties of in vivo and ex vivo malignant tissue (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 28% higher than normal). We report the one-pole Cole-Cole parameters for ex vivo normal, malignant and cirrhotic liver tissue in this frequency range. We observe that wideband dielectric properties of in vivo liver tissue are different from the wideband dielectric properties of ex vivo liver tissue, and that the in vivo data cannot be represented in terms of a Cole-Cole model. Further work is needed to uncover the mechanisms responsible for the observed wideband trends in the in vivo liver data.

(1)H magnetic resonance spectroscopy of preinvasive and invasive cervical cancer: in vivo-ex vivo profiles and effect of tumor load.

PubMed

Mahon, Marrita M; Cox, I Jane; Dina, Roberto; Soutter, W Patrick; McIndoe, G Angus; Williams, Andreanna D; deSouza, Nandita M

2004-03-01

To compare in vivo (1)H magnetic resonance (MR) spectra of preinvasive and invasive cervical lesions with ex vivo magic angle spinning (MAS) spectra of intact biopsies from the same subjects and to establish the effects of tumor load in the tissue sampled on the findings. A total of 51 subjects (nine with normal cervix, 10 with cervical intraepithelial neoplasia [CIN], and 32 with cervical cancer) underwent endovaginal MR at 1.5 T. Single-voxel (3.4 cm(3)) (1)H MR spectra were acquired and voxel tumor load was calculated (tumor volume within voxel as a percentage of voxel volume). Resonances from triglycerides -CH(2) and -CH(3) and choline-containing compounds (Cho) were correlated with voxel tumor load. Biopsies analyzed by (1)H MAS-MR spectroscopy (MRS) had metabolite levels correlated with tumor load in the sample at histology. In vivo studies detected Cho in normal, CIN, and cancer patients with no significant differences in levels (P = 0.93); levels were independent of voxel tumor load. Triglyceride -CH(2) and -CH(3) signals in-phase with Cho were present in 77% and 29%, respectively, of cancer subjects (but not in normal women or those with CIN), but did not correlate with voxel tumor load. Ex vivo cancer biopsies showed levels of triglycerides -CH(2) and -CH(3) and of Cho that were significantly greater than in normal or CIN biopsies (P < 0.05); levels were independent of the tumor load in the sample. The presence of -CH(2) in vivo predicted the presence of cancer with a sensitivity and specificity of 77.4% and 93.8% respectively, positive (PPV) and negative (NPV) predictive values were 96% and 68.2%; for -CH(2) ex vivo, sensitivity was 100%; specificity, 69%; PPV, 82%; and NPV, 100%. Elevated lipid levels are detected by MRS in vivo and ex vivo in cervical cancer and are independent of tumor load in the volume of tissue sampled. Copyright 2004 Wiley-Liss, Inc.

Evaluation of nanoformulated therapeutics in an ex-vivo bovine corneal irritation model.

PubMed

Bhasker, Sriramoju; Kislay, Roy; Rupinder, Kanwar K; Jagat, Kanwar R

2015-08-01

To determine the internalization and protective effects of potential ophthalmic formulations and nanoformulated natural proteins in ex-vivo bovine corneal alkali burn model. The bovine cornea obtained were subjected to the 0.5N NaOH insult that induced alkali burn and inflammation as observed in the in vivo situation. The toxic effects of the nanoformulation were evaluated in the normal and insult induced cornea using histological analysis. Internalization studies were carried out using in vivo imaging and analysis (IVIS, PerkinElmer, USA). The nanoformulations employed in this study showed no obvious changes in the integrity of the cornea. Further, improvements in the light transmittance and reduced inflammation were observed. The IVIS showed a dose dependant increase in the uptake of the nanoformulations with time. The nanoformulated bovine lactoferrin and SurR9-C84A (SR9) proteins evaluated in the ex vivo bovine corneal irritation model is the first of its kind, and we report here the non-toxic and therapeutic potential of these formulations for topical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hepatic radiofrequency ablation: in vivo and ex vivo comparisons of 15-gauge (G) and 17-G internally cooled electrodes

PubMed Central

Song, K D; Park, H J; Cha, D I; Kang, T W; Lee, J; Moon, J Y; Rhim, H

2015-01-01

Objective: To compare the performance of the 15-G internally cooled electrode with that of the conventional 17-G internally cooled electrode. Methods: A total of 40 (20 for each electrode) and 20 ablation zones (10 for each electrode) were made in extracted bovine livers and in in vivo porcine livers, respectively. Technical parameters, three dimensions [long-axis diameter (Dl), vertical-axis diameter (Dv) and short-axis diameter (Ds)], volume and the circularity (Ds/Dl) of the ablation zone were compared. Results: The total delivered energy was higher in the 15-G group than in the 17-G group in both ex vivo and in vivo studies (8.78 ± 1.06 vs 7.70 ± 0.98 kcal, p = 0.033; 11.20 ± 1.13 vs 8.49 ± 0.35 kcal, p = 0.001, respectively). The three dimensions of the ablation zone had a tendency to be larger in the 15-G group than in the 17-G group in both studies. The ablation volume was larger in the 15-G group than in the 17-G group in both ex vivo and in vivo studies (29.61 ± 7.10 vs 23.86 ± 3.82 cm3, p = 0.015; 10.26 ± 2.28 vs 7.79 ± 1.68 cm3, p = 0.028, respectively). The circularity of ablation zone was not significantly different in both the studies. Conclusion: The size of ablation zone was larger in the 15-G internally cooled electrode than in the 17-G electrode in both ex vivo and in vivo studies. Advances in knowledge: Radiofrequency ablation of hepatic tumours using 15-G electrode is useful to create larger ablation zones. PMID:25882688

Functional characterisation and permeation studies of lyophilised thiolated chitosan xerogels for buccal delivery of insulin.

PubMed

Boateng, Joshua S; Mitchell, John C; Pawar, Harshavardhan; Ayensu, Isaac

2014-01-01

Stable and mucoadhesive, lyophilised, thiolated chitosan xerogels, loaded with insulin for buccal mucosa deliv- ery, in place of the currently used parenteral route have been developed. The xerogels were backed with impervious ethyl- cellulose laminate to ensure unidirectional release and also loaded with enzyme inhibitor to enhance insulin permeability across the buccal mucosa. Characterisation of xerogels using(1) HNMR confirmed the degree of deacetylation of the syn- thesised thiolated chitosan. The amount of thiol groups immobilised on the modified chitosan was quantified by Ellman's reaction and molecular weight monitored by gel permeation chromatography. The stability of the secondary structure of insulin was examined by attenuated total reflectance Fourier transform infra-red spectroscopy and circular dichroism. In vitro and ex vivo permeation studies were undertaken by using EpiOral ™ and sheep buccal membrane respectively. Insu- lin released from thiolated chitosan xerogels, loaded with aprotinin (enzyme inhibitor and permeation enhancer) showed a 1.7-fold increase in permeation through EpiOral ™ buccal tissue construct compared to the pure drug. However, permea- tion was decreased for xerogels containing the enzyme inhibitor glutathione. Further, aprotinin containing xerogels en- hanced insulin permeation through sheep buccal membrane and demonstrated good linear correlation with the permeation data from the EpiOral ™ study. The results show the potential application of lyoph ilised thiolated chitosan xerogels con- taining aprotinin with improved mucoadhesion, penetration enhancing and enzyme inhibition characteristics for buccal mucosa delivery of macromolecules such as insulin.

Susceptibility of anthocyanins to ex vivo degradation in human saliva

PubMed Central

Kamonpatana, Kom; Giusti, M. Mónica; Chitchumroonchokchai, Chureeporn; MorenoCruz, Maria; Riedl, Ken M.; Kumar, Purnima; Failla, Mark L.

2013-01-01

Some fruits and their anthocyanin-rich extracts have been reported to exhibit chemopreventive activity in the oral cavity. Insights regarding oral metabolism of anthocyanins remain limited. Anthocyanin-rich extracts from blueberry, chokeberry, black raspberry, red grape, and strawberry were incubated ex vivo with human saliva from 14 healthy subjects. All anthocyanins were partially degraded in saliva. Degradation of chokeberry anthocyanins in saliva was temperature dependent and decreased by heating saliva to 80 °C and after removal of cells. Glycosides of delphinidin and petunidin were more susceptible to degradation than those of cyanidin, pelargonidin, peonidin and malvidin in both intact and artificial saliva. Stability of di- and tri-saccharide conjugates of anthocyanidins slightly, but significantly, exceeded that of monosaccharide compounds. Ex vivo degradation of anthocyanins in saliva was significantly decreased after oral rinsing with antibacterial chlorhexidine. These results suggest that anthocyanin degradation in the mouth is structure-dependent and largely mediated by oral microbiota. PMID:22868153

Evaluation of the In Vivo and Ex Vivo Binding of Novel BC1 Cannabinoid Receptor Radiotracers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, A.; Gatley, J.; Gifford, A.

The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with amore » half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice. Using an approach based on incubation of brain slices prepared from mice given intravenous [131I]AM2233 in either the presence or absence of AM2233 (unlabelled) it was possible to demonstrate a significant AM2233-displacable binding in the Swiss-Webster mice. Future studies will determine if this assay is appropriate for identifying the unknown binding site for AM2233 in the CB1 knockout mice.« less

[A new technique for ensuring negative surgical margins during partial nephrectomy: the ex vivo ultrasound control].

PubMed

Desmonts, A; Tillou, X; Le Gal, S; Secco, M; Orczyk, C; Bensadoun, H; Doerfler, A

2013-10-01

To evaluate the feasibility and the efficiency of intraoperative ex vivo ultrasound of resection margins in patients undergoing partial nephrectomy by urologist. Patients undergoing partial nephrectomy from July 2010 to November 2012 for T1-T2 renal tumors were included in analysis. Tumor margin status was immediately determined by ex vivo ultrasound done by the surgeon himself. Results were compared with margin status on definitive pathological evaluation. A total of 26 men and 15 women with a median age of 61 (30-82) years old were included in analysis. Intraoperative ex vivo ultrasound revealed negative surgical margins in 38 cases and positive margins in two. Final pathological results revealed negative margins in all except one case. Ultrasound sensitivity and specificity were 100% and 97%, respectively. Mean ultrasound duration was 1minute±1. Mean tumor and margin sizes were 3.4±1.8cm and 2.38±1.76mm, respectively. Intraoperative ex vivo ultrasound of resection margins in patients undergoing partial nephrectomy by a urologist seemed to be feasible, efficient and easy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Intraoperative diagnosis of nonpigmented nail tumours with ex vivo fluorescence confocal microscopy: 10 cases.

PubMed

Debarbieux, S; Gaspar, R; Depaepe, L; Dalle, S; Balme, B; Thomas, L

2015-04-01

Ex vivo fluorescence confocal microscopy (FCM) permits real-time imaging of freshly excised skin tissues. Its usefulness as a time-sparing alternative to frozen sections in Mohs surgery of basal cell carcinoma is well documented. The purpose of this study was to describe the ex vivo FCM features of a series of benign and malignant nonpigmented tumours of the nail unit, and to correlate them with conventional histopathology. Nail apparatus tumours from 10 patients were imaged during surgical exploration using ex vivo FCM after immersion in acridine orange. Confocal mosaics of the freshly performed biopsies were evaluated in real time and retrospectively compared with haematoxylin and eosin sections. Our series included two invasive epithelial tumours (Group 1), four in situ or minimally invasive squamous cell carcinomas (SCC) (Group 2), three benign epithelial tumours (Group 3) and one nodular melanoma (Group 4). The correlation was excellent for malignant epithelial tumours exhibiting marked cytological and architectural atypias (Bowen disease, invasive SCC and onycholemmal carcinoma). Onychomatricomas exhibited a very peculiar aspect with densely cellular papillae. The correlation was less favourable for minimally invasive well-differentiated SCCs with slight cytological atypias. The correlation was poor for our case of amelanotic invasive subungual melanoma. Ex vivo FCM could be a useful tool to shorten management of nonpigmented nail tumours: in the case of a malignant tumour, it could indeed lead to performing wide excision during the same surgical procedure and possibly assessing the surgical margins. © 2014 British Association of Dermatologists.

21 CFR 184.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium acid tartrate. 184.1077 Section 184.1077... Listing of Specific Substances Affirmed as GRAS § 184.1077 Potassium acid tartrate. (a) Potassium acid tartrate (C4H5KO6, CAS Reg. No. 868-14-4) is the potassium acid salt of l−(+)−tartaric acid and is also...

21 CFR 184.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium acid tartrate. 184.1077 Section 184.1077... GRAS § 184.1077 Potassium acid tartrate. (a) Potassium acid tartrate (C4H5KO6, CAS Reg. No. 868-14-4) is the potassium acid salt of l−(+)−tartaric acid and is also called potassium bitartrate or cream of...

21 CFR 184.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium acid tartrate. 184.1077 Section 184.1077... Listing of Specific Substances Affirmed as GRAS § 184.1077 Potassium acid tartrate. (a) Potassium acid tartrate (C4H5KO6, CAS Reg. No. 868-14-4) is the potassium acid salt of l−(+)−tartaric acid and is also...

Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin.

PubMed

Ferris, Donald R; Satoh, Akira; Mandefro, Berhan; Cummings, Gillian M; Gardiner, David M; Rugg, Elizabeth L

2010-10-01

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration. © 2010 The Authors. Journal compilation © 2010 Japanese Society of Developmental Biologists.

Evaluation of an ex vivo murine local lymph node assay: multiple endpoint comparison.

PubMed

Piccotti, Joseph R; Knight, Stephanie A; Gillhouse, Kimberly; Lagattuta, Mark S; Bleavins, Michael R

2006-01-01

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity. Copyright 2006 John Wiley & Sons, Ltd.

Microemulsion-Based Mucoadhesive Buccal Wafers: Wafer Formation, In Vitro Release, and Ex Vivo Evaluation.

PubMed

Pham, Minh Nguyet; Van Vo, Toi; Tran, Van-Thanh; Tran, Phuong Ha-Lien; Tran, Thao Truong-Dinh

2017-10-01

Microemulsion has the potentials to enhance dissolution as well as facilitate absorption and permeation of poorly water-soluble drugs through biological membranes. However, its application to govern a controlled release buccal delivery for local treatment has not been discovered. The aim of this study is to develop microemulsion-based mucoadhesive wafers for buccal delivery based on an incorporation of the microemulsion with mucoadhesive agents and mannitol. Ratio of oil to surfactant to water in the microemulsion significantly impacted quality of the wafers. Furthermore, the combination of carbopol and mannitol played a key role in forming the desired buccal wafers. The addition of an extra 50% of water to the formulation was suitable for wafer formation by freeze-drying, which affected the appearance and distribution of carbopol in the wafers. The amount of carbopol was critical for the enhancement of mucoadhesive properties and the sustained drug release patterns. Release study presented a significant improvement of the drug release profile following sustained release for 6 h. Ex vivo mucoadhesive studies provided decisive evidence to the increased retention time of wafers along with the increased carbopol content. The success of this study indicates an encouraging strategy to formulate a controlled drug delivery system by incorporating microemulsions into mucoadhesive wafers.

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium potassium tartrate. 184.1804 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is...

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium potassium tartrate. 184.1804 Section 184... as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is also called the Rochelle...

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium potassium tartrate. 184.1804 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is...

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium potassium tartrate. 184.1804 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is...

Enhanced stability and permeation potential of nanoemulsion containing sefsol-218 oil for topical delivery of amphotericin B.

PubMed

Hussain, Afzal; Samad, Abdus; Singh, Sandeep Kumar; Ahsan, Mohd Neyaz; Faruk, Abdul; Ahmed, Farhan Jalees

2015-05-01

To characterize the enhanced stability and permeation potential of amphotericin B nanoemulsion comprising sefsol-218 oil at varying pH and temperature of aqueous continuous phase. Several batches of amphotericin B loaded nanoemulsion were prepared and evaluated for their physical and chemical stability at different pH and temperature. Also, a comparative study of ex vivo drug permeation across the albino rat skin was investigated with commercial Fungisome® and drug solution at 37 °C for 24 h. The extent of drug penetrated through the rat skin was thereby evaluated using the confocal laser scanning microscopy (CLSM). The optimized nanoemulsion demonstrated the highest flux rate 17.85 ± 0.5 µg/cm(2)/h than drug solution (5.37 ± 0.01 µg/cm(2)/h) and Fungisome® (7.97 ± 0.01 µg/cm(2)/h). Ex vivo drug penetration mechanism from the developed formulations at pH 6.8 and pH 7.4 of aqueous phase pH using the CLSM revealed enhanced penetration. Ex vivo drug penetration studies of developed formulation comprising of CLSM revealed enhanced penetration in aqueous phase at pH 6.8 and 7.4. The aggregation behavior of nanoemulsion at both the pH was found to be minimum and non-nephrotoxic. The stability of amphotericin B was obtained in terms of pH, optical density, globular size, polydispersity index and zeta potential value at different temperature for 90 days. The slowest drug degradation was observed in aqueous phase at pH 7.4 with shelf life 20.03-folds higher when stored at 4 °C (3.8 years) and 5-fold higher at 25 °C (0.951 years) than at 40 °C. The combined results suggested that nanoemulsion may hold an alternative for enhanced and sustained topical delivery system for amphotericin B.

PGE2 suppresses NK activity in vivo directly and through adrenal hormones: Effects that cannot be reflected by ex-vivo assessment of NK cytotoxicity

PubMed Central

Meron, G.; Tishler, Y.; Shaashua, L.; Rosenne, E.; Levi, B.; Melamed, R.; Gotlieb, N.; Matzner, P.; Sorski, L.; Ben-Eliyahu, S.

2013-01-01

Surgery can suppress in vivo levels of NK cell cytotoxicity (NKCC) through various mechanisms, including catecholamine-, glucocorticoid (CORT)-, and prostaglandin (PG)-mediated responses. However, PGs are synthesized locally following tissue damage, driving proinflammatory and CORT responses, while their systemic levels are often unaffected. Thus, we herein studied the role of adrenal factors in mediating in vivo effects of PGs on NKCC, using adrenalectomized and sham-operated F344 rats subjected to surgery or PGE2 administration. In vivo and ex-vivo approaches were employed, based on intravenous administration of the NK-sensitive MADB106 tumor line, and based on ex-vivo assessment of YAC-1 and MADB106 target-line lysis. Additionally, in vitro studies assessed the kinetics of the impact of epinephrine, CORT, and PGE2 on NKCC. The results indicated that suppression of NKCC by epinephrine and PGE2 are short lasting, and cannot be evident when these compounds are removed from the in vitro assay milieu, or in the context of ex-vivo assessment of NKCC. In contrast, the effects of CORT are long-lasting and are reflected in both conditions even after its removal. Marginating-pulmonary NKCC was less susceptible to suppression than circulating NKCC, when tested against the xenogeneic YAC-1 target line, but not against the syngeneic MADB106 line, which seems to involve different cytotoxicity mechanisms. Overall, these findings indicate that elevated systemic PG levels can directly suppress NKCC in vivo, but following laparotomy adrenal hormones mediate most of the effects of endogenously-released PGs. Additionally, the ex-vivo approach seems limited in reflecting the short-lasting NK-suppressive effects of catecholamines and PGs. PMID:23153554

PGE2 suppresses NK activity in vivo directly and through adrenal hormones: effects that cannot be reflected by ex vivo assessment of NK cytotoxicity.

PubMed

Meron, G; Tishler, Y; Shaashua, L; Rosenne, E; Levi, B; Melamed, R; Gotlieb, N; Matzner, P; Sorski, L; Ben-Eliyahu, S

2013-02-01

Surgery can suppress in vivo levels of NK cell cytotoxicity (NKCC) through various mechanisms, including catecholamine-, glucocorticoid (CORT)-, and prostaglandin (PG)-mediated responses. However, PGs are synthesized locally following tissue damage, driving proinflammatory and CORT responses, while their systemic levels are often unaffected. Thus, we herein studied the role of adrenal factors in mediating in vivo effects of PGs on NKCC, using adrenalectomized and sham-operated F344 rats subjected to surgery or PGE(2) administration. In vivo and ex vivo approaches were employed, based on intravenous administration of the NK-sensitive MADB106 tumor line, and based on ex vivo assessment of YAC-1 and MADB106 target-line lysis. Additionally, in vitro studies assessed the kinetics of the impact of epinephrine, CORT, and PGE(2) on NKCC. The results indicated that suppression of NKCC by epinephrine and PGE(2) are short lasting, and cannot be evident when these compounds are removed from the in vitro assay milieu, or in the context of ex vivo assessment of NKCC. In contrast, the effects of CORT are long-lasting and are reflected in both conditions even after its removal. Marginating-pulmonary NKCC was less susceptible to suppression than circulating NKCC, when tested against the xenogeneic YAC-1 target line, but not against the syngeneic MADB106 line, which seems to involve different cytotoxicity mechanisms. Overall, these findings indicate that elevated systemic PG levels can directly suppress NKCC in vivo, but following laparotomy adrenal hormones mediate most of the effects of endogenously-released PGs. Additionally, the ex vivo approach seems limited in reflecting the short-lasting NK-suppressive effects of catecholamines and PGs. Copyright © 2012 Elsevier Inc. All rights reserved.

Recent developments in skin mimic systems to predict transdermal permeation.

PubMed

Waters, Laura J

2015-01-01

In recent years there has been a drive to create experimental techniques that can facilitate the accurate and precise prediction of transdermal permeation without the use of in vivo studies. This review considers why permeation data is essential, provides a brief summary as to how skin acts as a natural barrier to permeation and discusses why in vivo studies are undesirable. This is followed by an in-depth discussion on the extensive range of alternative methods that have been developed in recent years. All of the major 'skin mimic systems' are considered including: in vitro models using synthetic membranes, mathematical models including quantitative structure-permeability relationships (QSPRs), human skin equivalents and chromatographic based methods. All of these model based systems are ideally trying to achieve the same end-point, namely a reliable in vitro-in vivo correlation, i.e. matching non-in vivo obtained data with that from human clinical trials. It is only by achieving this aim, that any new method of obtaining permeation data can be acknowledged as a potential replacement for animal studies, for the determination of transdermal permeation. In this review, the relevance and potential applicability of the various models systems will also be discussed.

Preparation of silica nanoparticles loaded with nootropics and their in vivo permeation through blood-brain barrier.

PubMed

Jampilek, Josef; Zaruba, Kamil; Oravec, Michal; Kunes, Martin; Babula, Petr; Ulbrich, Pavel; Brezaniova, Ingrid; Opatrilova, Radka; Triska, Jan; Suchy, Pavel

2015-01-01

The blood-brain barrier prevents the passage of many drugs that target the central nervous system. This paper presents the preparation and characterization of silica-based nanocarriers loaded with piracetam, pentoxifylline, and pyridoxine (drugs from the class of nootropics), which are designed to enhance the permeation of the drugs from the circulatory system through the blood-brain barrier. Their permeation was compared with non-nanoparticle drug substances (bulk materials) by means of an in vivo model of rat brain perfusion. The size and morphology of the nanoparticles were characterized by transmission electron microscopy. The content of the drug substances in silica-based nanocarriers was analysed by elemental analysis and UV spectrometry. Microscopic analysis of visualized silica nanocarriers in the perfused brain tissue was performed. The concentration of the drug substances in the tissue was determined by means of UHPLC-DAD/HRMS LTQ Orbitrap XL. It was found that the drug substances in silica-based nanocarriers permeated through the blood brain barrier to the brain tissue, whereas bulk materials were not detected in the brain.

Preparation of Silica Nanoparticles Loaded with Nootropics and Their In Vivo Permeation through Blood-Brain Barrier

PubMed Central

Zaruba, Kamil; Kunes, Martin; Ulbrich, Pavel; Brezaniova, Ingrid; Triska, Jan; Suchy, Pavel

2015-01-01

The blood-brain barrier prevents the passage of many drugs that target the central nervous system. This paper presents the preparation and characterization of silica-based nanocarriers loaded with piracetam, pentoxifylline, and pyridoxine (drugs from the class of nootropics), which are designed to enhance the permeation of the drugs from the circulatory system through the blood-brain barrier. Their permeation was compared with non-nanoparticle drug substances (bulk materials) by means of an in vivo model of rat brain perfusion. The size and morphology of the nanoparticles were characterized by transmission electron microscopy. The content of the drug substances in silica-based nanocarriers was analysed by elemental analysis and UV spectrometry. Microscopic analysis of visualized silica nanocarriers in the perfused brain tissue was performed. The concentration of the drug substances in the tissue was determined by means of UHPLC-DAD/HRMS LTQ Orbitrap XL. It was found that the drug substances in silica-based nanocarriers permeated through the blood brain barrier to the brain tissue, whereas bulk materials were not detected in the brain. PMID:26075264

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

PubMed Central

Perrin-Terrin, Anne-Sophie; Jeton, Florine; Pichon, Aurelien; Frugière, Alain; Richalet, Jean-Paul; Bodineau, Laurence; Voituron, Nicolas

2016-01-01

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive. PMID:27167092

Inhibition of ex vivo proinflammatory cytokine secretion in fatal Mycobacterium tuberculosis infection.

PubMed Central

Friedland, J S; Hartley, J C; Hartley, C G; Shattock, R J; Griffin, G E

1995-01-01

Tuberculosis is characterized by fever, weight loss, a prolonged acute-phase protein response and granuloma formation. These characteristics may partly be due to action of proinflammatory cytokines tumour necrosis factor (TNF), IL-6 and IL-8. We investigated plasma concentrations of these cytokines before and after ex vivo lipopolysaccharide stimulation of whole blood leucocytes from 41 Zambian patients with tuberculosis, 32 of whom were also HIV+. Although patients had a reduced weight, were more anaemic and had higher erythrocyte sedimentation rate compared with controls (all P < 0.0005), clinical and laboratory measurements of disease state were similar in those who died and survivors. In contrast, plasma IL-6 and IL-8 concentrations were higher in patients who died (P < 0.05). There was no detectable cytokine mRNA in unstimulated leucocytes. There was reduced secretion of TNF (P < 0.005 at 2 h), IL-6 (P < 0.005 at 8 h) and IL-8 (P < 0.005 at 24 h) after ex vivo stimulation of whole blood leucocytes from patients who died compared with survivors. This was partly due to a soluble inhibitory factor present in plasma. The only additional effect of concurrent infection by HIV with Myco. tuberculosis was decreased IL-6 secretion following ex vivo stimulation of leucocytes. Reduced proinflammatory cytokine release may represent a critical impairment of host immune defences important in determining outcome in tuberculosis. PMID:7743661

The protective effect of dexmedetomidine in a rat ex vivo lung model of ischemia-reperfusion injury.

PubMed

Zhou, Yan; Zhou, Xinqiao; Zhou, Wenjuan; Pang, Qingfeng; Wang, Zhiping

2018-01-01

To investigate the effect of dexmedetomidine (Dex) in a rat ex vivo lung model of ischemia-reperfusion injury. An IL-2 ex vivo lung perfusion system was used to establish a rat ex vivo lung model of ischemia-reperfusion injury. Drugs were added to the perfusion solution for reperfusion. Lung injury was assessed by histopathological changes, airway pressure (Res), lung compliance (Compl), perfusion flow (Flow), pulmonary venous oxygen partial pressure (PaO2), and lung wet/dry (W/D) weight ratio. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), 78 kDa glucose-regulated protein (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) were measured, respectively. The introduction of Dex attenuated the post-ischemia-reperfusion lung damage and MDA level, improved lung histology, W/D ratio, lung injury scores and SOD activity. Decreased mRNA and protein levels of GRP78 and CHOP compared with the IR group were observed after Dex treatment. The effect of Dex was dosage-dependence and a high dose of Dex (10 nM) was shown to confer the strongest protective effect against lung damage (P<0.05). Yohimbine, an α2 receptor antagonist, significantly reversed the protective effect of Dex in lung tissues (P<0.05). Dex reduced ischemia-reperfusion injury in rat ex vivo lungs.

21 CFR 582.1077 - Potassium acid tartrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Additives § 582.1077 Potassium acid tartrate. (a) Product. Potassium acid tartrate. (b) Conditions of use. This substance is generally recognized as safe when used in accordance with good manufacturing or...

Ex vivo instability of glycated albumin: A role for autoxidative glycation.

PubMed

Jeffs, Joshua W; Ferdosi, Shadi; Yassine, Hussein N; Borges, Chad R

2017-09-01

Ex vivo protein modifications occur within plasma and serum (P/S) samples due to prolonged exposure to the thawed state-which includes temperatures above -30 °C. Herein, the ex vivo glycation of human serum albumin from healthy and diabetic subjects was monitored in P/S samples stored for hours to months at -80 °C, -20 °C, and room temperature, as well as in samples subjected to multiple freeze-thaw cycles, incubated at different surface area-to-volume ratios or under different atmospheric compositions. A simple dilute-and-shoot method utilizing trap-and-elute LC-ESI-MS was employed to determine the relative abundances of the glycated forms of albumin-including forms of albumin bearing more than one glucose molecule. Significant increases in glycated albumin were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin-suggesting a role for oxidative glycation in the ex vivo glycation of albumin. Copyright © 2017 Elsevier Inc. All rights reserved.

An Ex Vivo Comparison of 2 Cyanoacrylate Skin Protectants.

PubMed

Gibson, Daniel J

The purpose of these experiments was to compare 2 commercially available skin protectants with different chemical compositions. Two materially different skin protectants were applied to ex vivo pig skin, subjected to stresses, and the resulting skin was observed and analyzed. Using ex vivo pig skin, we sought to better understand the physical differences between a cyanoacrylate-based and a mixed cyanoacrylate/acrylic polymer-based skin protectant. A combination of imaging techniques and microscopic analyses was used to observe and quantify differences in layer thickness and the degree of steadfastness of the layers to liquid stresses. The experiments revealed that the solely cyanoacrylate-based protectant created a layer that was, on average, 5.1 times thicker than the mixed polymer product (p= 1.8 × 10). Observation via electron microscopy also revealed that the extent of coverage varied between the 2 products. In a final experiment, we observed that the mixed polymer product maintained a high degree of adhesiveness, which led to the removal of sheets of epithelium upon gentle blotting. The experiments revealed that while the 2 skin protectants share a common ingredient, both the quantity of that ingredient and the inclusion of other materials in one of them lead to substantially different properties when tested in the research setting.

In vivo and ex vivo EPR detection of spin-labelled ovalbumin in mice.

PubMed

Abramović, Zrinka; Brgles, Marija; Habjanec, Lidija; Tomasić, Jelka; Sentjurc, Marjeta; Frkanec, Ruza

2010-10-01

In this study, spin-labelled ovalbumin (SL-OVA), free or entrapped in liposomes, was administered to mice subcutaneously (s.c.) or intravenously (i.v.) with the aim to determine the conditions for pharmacokinetic studies of spin-labelled proteins by EPR and to measure the time course of SL-OVA distribution in vivo in live mice and ex vivo in isolated organs. Upon s.c. administration, the decay of the EPR signal was followed for 60min at the site of application using an L-band EPR spectrometer. Within this time period, the signal of free SL-OVA was diminished by about 70%. It was estimated with the help of the oxidizing agent K(3)[(FeCN)(6)] that approximately 30% was a consequence of the spin label reduction to EPR non-visible hydroxylamine and about 40% was due to the SL-OVA elimination from the site of measurement. For liposome encapsulated SL-OVA, the intensity diminished only by approx. 40% in the same period, indicating that liposomes successfully protect the protein from reduction. EPR signal could not be detected directly over live mouse organs within 60min after s.c. application of SL-OVA. With the available L-band EPR spectrometer, the measurements at the site of s.c. application are possible if the amount of SL-OVA applied to a mouse is more than 3mg. For the pharmacokinetic studies of the protein distribution in organs after s.c. or i.v. injection the concentration of the spin-labelled protein should be more than 0.5mmol/kg. After i.v. administration, only ex vivo measurements were possible using an X-band EPR spectrometer, since the total amount of SL-OVA was not sufficient for in vivo detection and also because of rapid reduction of nitroxide. After 2min, the protein was preferentially distributed to liver and, to a smaller extent, to spleen.

An ex vivo human skin model for studying skin barrier repair.

PubMed

Danso, Mogbekeloluwa O; Berkers, Tineke; Mieremet, Arnout; Hausil, Farzia; Bouwstra, Joke A

2015-01-01

In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32 °C and 37 °C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37 °C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32 °C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37 °C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32 °C and 37 °C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37 °C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32 °C. Potentially, this model can be used for testing formulations for skin barrier repair. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ex Vivo Activity of Endoperoxide Antimalarials, Including Artemisone and Arterolane, against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

DTIC Science & Technology

2014-10-01

OCT 2014 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Ex Vivo Activity of Endoperoxide Antimalarials , Including Artemisone...Prescribed by ANSI Std Z39-18 Ex Vivo Activity of Endoperoxide Antimalarials , Including Artemisone and Arterolane, against Multidrug-Resistant...potent antimalarial activity (2, 3). Despite having a rapid mecha- nism of action, artemisinin resistance eventually emerged and was first detected

Development and characterization of an ex-vivo brain slice culture model of chronic wasting disease

USDA-ARS?s Scientific Manuscript database

Prion diseases have long incubation times in vivo, therefore, modeling the diseases ex-vivo will advance the development of rationale-based therapeutic strategies. An organotypic slice culture assay (POSCA) was recently developed for scrapie prions by inoculating mouse cerebellar brain slices with R...

Determination of lamivudine and zidovudine permeability using a different ex vivo method in Franz cells.

PubMed

Dezani, André Bersani; Pereira, Thaisa Marinho; Caffaro, Arthur Massabki; Reis, Juliana Mazza; Serra, Cristina Helena Dos Reis

2013-01-01

The major processes that control the absorption of orally administered drugs are dissolution and gastrointestinal permeation. These processes depend on two main properties: solubility and permeability. Based on these characteristics, the Biopharmaceutical Classification System (BCS) was proposed as a tool to assist in biowaiver and bioavailability prediction of drugs. The purpose of the present study was to evaluate the permeability of lamivudine (3TC) and zidovudine (AZT) using a different ex vivo method in Franz cells. A segment of jejunum was inserted in a Franz cells apparatus, in order to assess drug permeability in the apical-basolateral (A-B) and basolateral-apical (B-A) directions. Each drug was added to the donor chamber, collected from the acceptor chamber and analyzed by HPLC. Fluorescein (FLU) and metoprolol (METO) were used as low and high permeability markers, respectively. The apparent permeability (Papp) results for the A-B direction were: Papp FLU A-B=0.54×10(-4)cm·s(-1), Papp METO A-B=7.99×10(-4)cm·s(-1), Papp 3TC A-B=4.58×10(-4)cm·s(-1) and Papp AZT A-B=5.34×10(-4)cm·s(-1). For the B-A direction, the Papp results were: Papp FLU B-A=0.56×10(-4)cm·s(-1), Papp METO B-A=0.25×10(-4)cm·s(-1), Papp 3TC B-A=0.24×10(-4)cm·s(-1) and Papp AZT B-A=0.19×10(-4)cm·s(-1). For the A-B direction, the Papp results of fluorescein and metoprolol show low and high permeability, respectively, indicating that the membranes were appropriate for permeability studies. For the A-B direction, the Papp results of 3TC and AZT suggest that these antiretroviral drugs have permeability values close to metoprolol. Nevertheless, for the B-A direction the Papp results do not suggest efflux mechanism for any of the drugs. Thereby, the different ex vivo methods using Franz cells can be successfully applied in drug permeability studies, in particular for drug biopharmaceutical classification. Copyright © 2013 Elsevier Inc. All rights reserved.

Long-term ex vivo and in vivo monitoring of tumor progression by using dual luciferases.

PubMed

Morita, Naoki; Haga, Sanae; Ohmiya, Yoshihiro; Ozaki, Michitaka

2016-03-15

We propose a new concept of tumor progression monitoring using dual luciferases in living animals to reduce stress for small animals and the cost of luciferin. The secreted Cypridina luciferase (CLuc) was used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase was used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Thus, the new monitoring systems that use dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals. Copyright © 2015 Elsevier Inc. All rights reserved.

In situ measurement of tritium permeation through stainless steel

NASA Astrophysics Data System (ADS)

Luscher, Walter G.; Senor, David J.; Clayton, Kevin K.; Longhurst, Glen R.

2013-06-01

The TMIST-2 irradiation experiment was conducted in the Advanced Test Reactor at Idaho National Laboratory to evaluate tritium permeation through Type 316 stainless steel (316 SS). The interior of a 316 SS seamless tube specimen was exposed to a 4He carrier gas mixed with a specified quantity of tritium (T2) to yield partial pressures of 0.1, 5, and 50 Pa at 292 °C and 330 °C. In situ tritium permeation measurements were made by passing a He-Ne sweep gas over the outer surface of the specimen to carry the permeated tritium to a bubbler column for liquid scintillation counting. Results from in situ permeation measurements were compared with predictions based on an ex-reactor permeation correlation in the literature. In situ permeation data were also used to derive an in-reactor permeation correlation as a function of temperature and pressure over the ranges considered in this study. In addition, the triton recoil contribution to tritium permeation, which results from the transmutation of 3He to T, was also evaluated by introducing a 4He carrier gas mixed with 3He at a partial pressure of 1013 Pa at 330 °C. Less than 3% of the tritium resulting from 3He transmutation contributed to tritium permeation.

Ex-vivo expanded umbilical cord blood stem cells retain capacity for myocardial regeneration.

PubMed

Schlechta, Bernhard; Wiedemann, Dominik; Kittinger, Clemens; Jandrositz, Anita; Bonaros, Nikolaos E; Huber, Johannes C; Preisegger, Karl-Heinz; Kocher, Alfred A

2010-01-01

Umbilical cord blood (UCB) is a source of human hematopoietic precursor cells (HPCs), a stem cell (SC) type that has been used in several trials for myocardial repair. A certain minimal number of cells is required for measurable regeneration and a major challenge of SC-based regenerative therapy constitutes ex-vivo expansion of the primitive cell compartment. The aim of this study was to investigate the ex-vivo expansion potential of UCB-derived HPCs and the ability of these expanded cells to migrate to the site of damage and improve ventricular function in a rodent model of myocardial infarction (MI). UCB-derived HPCs, defined by coexpression of CD133 and CD34, were expanded using various cytokine combinations. MI was induced by left anterior descending artery ligation in nude rats. Cells were injected intravenously 2 days after infarction. The combination of SC factor, thrombopoietin, flt3-ligand and interleukin-6 was found to be the most effective for inducing proliferation of HPCs. The migratory capacity of expanded HPCs was similar to that of non-expanded HPCs and improvement of ejection fraction was significant in both groups, with a relative increase of >60%. UCB-derived HPCs can be reproducibly expanded ex-vivo and retain their potential to improve cardiac function post-MI. (Circ J 2010; 74: 188 - 194).

Terbinafine hydrochloride nanovesicular gel: In vitro characterization, ex vivo permeation and clinical investigation.

PubMed

AbdelSamie, Sara M; Kamel, Amany O; Sammour, Omaima A; Ibrahim, Shady M

2016-06-10

In this work, nanovesicular chitosan gels were prepared for dermal delivery of terbinafine hydrochloride (TBN HCl). Ethosomes and vesicles containing different types of penetration enhancers (PEs) viz. Terpenes (cineole and limonene), labrasol and transcutol were developed. The prepared vesicles were evaluated for physical characteristics as well as skin interaction. The selected vesicles were incorporated into chitosan gel. An in vivo animal study was done on rat induced superficial Candida infection model. Moreover, randomized double blind clinical study was done on patients to compare the effect of the selected nanovesicular gel against the market product. Results showed the formation of nearly spherical, mostly deformable vesicular systems with size range of 95.5-530nm, zeta potential range of -0.1 to 15mV and entrapment efficiency range of 20-96.7%. Penetration enhancer vesicles (PEVs) prepared with 4% limonene (ELI4) showed the highest percent of drug deposition in the skin (53%) and the highest local accumulation efficiency value (35.3). In vivo animal study showed that the lowest fungal burden produced with ELI4 chitosan gel. Clinical studies showed cure rate of 86% within 7days treatment in case of limonene nanovesicular gel compared to 20% for market product (Lamisil® cream). Copyright © 2016 Elsevier B.V. All rights reserved.

'En face' ex vivo reflectance confocal microscopy to help the surgery of basal cell carcinoma of the eyelid.

PubMed

Espinasse, Marine; Cinotti, Elisa; Grivet, Damien; Labeille, Bruno; Prade, Virginie; Douchet, Catherine; Cambazard, Frédéric; Thuret, Gilles; Gain, Philippe; Perrot, Jean Luc

2017-07-01

Ex vivo confocal microscopy is a recent imaging technique for the perioperative control of skin tumour margins. Up to date, it has been used in the fluorescence mode and with vertical sections of the specimen margins. The aim of this study was to evaluate its use in the reflectance mode and with a horizontal ('en face') scanning of the surgical specimen in a series of basal cell carcinoma of the eyelid. Prospective consecutive cohort study was performed at the University Hospital of Saint-Etienne, France. Forty-one patients with 42 basal cell carcinoma of the eyelid participated in this study. Basal cell carcinomas were excised with a 2-mm-wide clinically safe margin. The surgical specimens were analysed under ex vivo confocal microscopy in the reflectance mode and with an en face scanning in order to control at a microscopic level if the margins were free from tumour invasion. Histopathogical examination was later performed in order to compare the results. Sensitivity and specificity of ex vivo confocal microscopy for the presence of tumour-free margins. Ex vivo confocal microscopy results were consistent with histopathology in all cases (tumour-free margins in 40 out of 42 samples; sensitivity and specificity of 100%). Ex vivo confocal microscopy in the reflectance mode with an 'en face' scanning can control tumour margins of eyelid basal cell carcinomas and optimize their surgical management. This procedure has the advantage on the fluorescent mode of not needing any contrast agent to examine the samples. © 2016 Royal Australian and New Zealand College of Ophthalmologists.

Microwave Ablation With a Triaxial Antenna: Results in ex vivo Bovine Liver

PubMed Central

Brace, Christopher L.; Laeseke, Paul F.; van der Weide, Daniel W.; Lee, Fred T.

2007-01-01

We apply a new triaxial antenna for microwave ablation procedures to an ex vivo bovine liver. The antenna consists of a coaxial monopole inserted through a biopsy needle positioned one quarter-wavelength from the antenna base. The insertion needle creates a triaxial structure, which enhances return loss more than 10 dB, maximizing energy transfer to the tissue while minimizing feed cable heating and invasiveness. Numerical electromagnetic and thermal simulations are used to optimize the antenna design and predict heating patterns. Numerical and ex vivo experimental results show that the lesion size depends strongly on ablation time and average input power, but not on peak power. Pulsing algorithms are also explored. We were able to measure a 3.8-cm lesion using 50 W for 7 min, which we believe to be the largest lesion reported thus far using a 17-gauge insertion needle. PMID:18079981

The effects of muscle contraction and recombinant osteocalcin on insulin sensitivity ex vivo.

PubMed

Levinger, I; Lin, X; Zhang, X; Brennan-Speranza, T C; Volpato, B; Hayes, A; Jerums, G; Seeman, E; McConell, G

2016-02-01

We tested whether GPRC6A, the putative receptor of undercarboxylated osteocalcin (ucOC), is present in mouse muscle and whether ucOC increases insulin sensitivity following ex vivo muscle contraction. GPPRC6A is expressed in mouse muscle and in the mouse myotubes from a cell line. ucOC potentiated the effect of ex vivo contraction on insulin sensitivity. Acute exercise increases skeletal muscle insulin sensitivity. In humans, exercise increases circulating ucOC, a hormone that increases insulin sensitivity in rodents. We tested whether GPRC6A, the putative receptor of ucOC, is present in mouse muscle and whether recombinant ucOC increases insulin sensitivity in both C2C12 myotubes and whole mouse muscle following ex vivo muscle contraction. Glucose uptake was examined in C2C12 myotubes that express GPRC6A following treatment with insulin alone or with insulin and increasing ucOC concentrations (0.3, 3, 10 and 30 ng/ml). In addition, glucose uptake, phosphorylated (p-)AKT and p-AS160 were examined ex vivo in extensor digitorum longus (EDL) dissected from C57BL/6J wild-type mice, at rest, following insulin alone, after muscle contraction followed by insulin and after muscle contraction followed by recombinant ucOC then insulin exposure. We observed protein expression of the likely receptor for ucOC, GPRC6A, in whole muscle sections and differentiated mouse myotubes. We observed reduced GPRC6A expression following siRNA transfection. ucOC significantly increased insulin-stimulated glucose uptake dose-dependently up to 10 ng/ml, in differentiated mouse C2C12 myotubes. Insulin increased EDL glucose uptake (∼30 %, p < 0.05) and p-AKT and p-AKT/AKT compared with rest (all p < 0.05). Contraction prior to insulin increased muscle glucose uptake (∼25 %, p < 0.05), p-AKT, p-AKT/AKT, p-AS160 and p-AS160/AS160 compared with contraction alone (all p < 0.05). ucOC after contraction increased insulin-stimulated muscle glucose uptake (∼12 % p < 0

A new ex vivo method for the study of nasal drops on ciliary function.

PubMed

Levrier, J; Molon-Noblot, S; Duval, D; Lloyd, K G

1989-01-01

Any pharmaceutical nasal preparation should respect the physiological function of the mucociliary transport system and should undergo testing to this effect. An experimental protocol has been developed using the guinea pig in order to assess the effects of commercial nasal drop preparations on mucociliary function. The method presented here consists of applying in vivo the test solution on the nasal respiratory epithelium. After a specified contact time and following rapid sacrifice of the animal, the mucosa is removed; the beating frequency of the cilia is then recorded ex vivo by micro-photo-oscillography. The method is sensitive to compounds known to diminish mucociliary function as sodium mercurothiolate inhibits ciliary movement of the nasal epithelium ex vivo. This inhibition of ciliary movement is long-lasting, although reversible. This method can be used to test the action of intranasally administered pharmaceutical preparations on mucociliary function. Commercially available solutions of the nasal vasoconstrictors tymazoline, fenoxazoline or oxymetazoline do not alter ciliary movement ex vivo at dose levels equal to or greater than those clinically utilized. ATP significantly enhances nasal ciliary frequency in instances where a low basal rate occurred. Thus, this method can be used for the testing of the maintenance of nasal ciliary function in the presence of compounds and preparations which will be applied into the nostrils. The advantages over previous techniques include a closer approach to the therapeutic utilization and the maintained physiological conditions of the mucosa during drug administration.

Multidimensional custom-made non-linear microscope: from ex-vivo to in-vivo imaging

NASA Astrophysics Data System (ADS)

Cicchi, R.; Sacconi, L.; Jasaitis, A.; O'Connor, R. P.; Massi, D.; Sestini, S.; de Giorgi, V.; Lotti, T.; Pavone, F. S.

2008-09-01

We have built a custom-made multidimensional non-linear microscope equipped with a combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging. The optical system was mounted on a vertical honeycomb breadboard in an upright configuration, using two galvo-mirrors relayed by two spherical mirrors as scanners. A double detection system working in non-descanning mode has allowed both photon counting and a proportional regime. This experimental setup offering high spatial (micrometric) and temporal (sub-nanosecond) resolution has been used to image both ex-vivo and in-vivo biological samples, including cells, tissues, and living animals. Multidimensional imaging was used to spectroscopically characterize human skin lesions, as malignant melanoma and naevi. Moreover, two-color detection of two photon excited fluorescence was applied to in-vivo imaging of living mice intact neocortex, as well as to induce neuronal microlesions by femtosecond laser burning. The presented applications demonstrate the capability of the instrument to be used in a wide range of biological and biomedical studies.

Expanding living kidney donor criteria with ex-vivo surgery for renal anomalies

PubMed Central

McGregor, Thomas B.; Rampersad, Christie; Patel, Premal

2016-01-01

Introduction: Renal transplantation remains the gold standard treatment for end-stage renal disease, with living donor kidneys providing the best outcomes in terms of allograft survival. As the number of patients on the waitlist continues to grow, solutions to expand the donor pool are ongoing. A paradigm shift in the eligibility of donors with renal anomalies has been looked at as a potential source to expand the living donor pool. We sought to determine how many patients presented with anatomic renal anomalies at our transplant centre and describe the ex-vivo surgical techniques used to render these kidneys suitable for transplantation. Methods: A retrospective review was performed of all patients referred for surgical suitability to undergo laparoscopic donor nephrectomy between January 2011 and January 2015. Patient charts were analyzed for demographic information, perioperative variables, urological histories, and postoperative outcomes. Results: 96 referrals were identified, of which 81 patients underwent laparoscopic donor nephrectomy. Of these patients, 11 (13.6%) were identified as having a renal anomaly that could potentially exclude them from the donation process. These anomalies included five patients with unilateral nephrolithiasis, four patients with large renal cysts (>4 cm diameter), one patient with an angiomyolipoma (AML) and one patient with a calyceal diverticulum filled with stones. A description of the ex-vivo surgical techniques used to correct these renal anomalies is provided. Conclusions: We have shown here that ex-vivo surgical techniques can safely and effectively help correct some of these renal anomalies to render these kidneys transplantable, helping to expand the living donor pool. PMID:27800047

Ex-vivo assessment and non-invasive in vivo imaging of internal hemorrhages in Aga2/+ mutant mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ermolayev, Vladimir; Cohrs, Christian M.; Mohajerani, Pouyan

Highlights: ► Aga2/+ mice, model for Osteogenesis imperfecta, have type I collagen mutation. ► Aga2/+ mice display both moderate and severe phenotypes lethal 6–11th postnatal. ► Internal hemorrhages studied in Aga2/+ vs. control mice at 6 and 9 days postnatal. ► Anatomical and functional findings in-vivo contrasted to the ex-vivo appearance. -- Abstract: Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1{sup Aga2/+}, animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks andmore » internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6–11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing.« less

Nondestructive tissue analysis for ex vivo and in vivo cancer diagnosis using a handheld mass spectrometry system

PubMed Central

Zhang, Jialing; Rector, John; Lin, John Q.; Young, Jonathan H.; Sans, Marta; Katta, Nitesh; Giese, Noah; Yu, Wendong; Nagi, Chandandeep; Suliburk, James; Liu, Jinsong; Bensussan, Alena; DeHoog, Rachel J.; Garza, Kyana Y.; Ludolph, Benjamin; Sorace, Anna G.; Syed, Anum; Zahedivash, Aydin; Milner, Thomas E.; Eberlin, Livia S.

2018-01-01

Conventional methods for histopathologic tissue diagnosis are labor- and time-intensive and can delay decision-making during diagnostic and therapeutic procedures. We report the development of an automated and biocompatible handheld mass spectrometry device for rapid and nondestructive diagnosis of human cancer tissues. The device, named MasSpec Pen, enables controlled and automated delivery of a discrete water droplet to a tissue surface for efficient extraction of biomolecules. We used the MasSpec Pen for ex vivo molecular analysis of 20 human cancer thin tissue sections and 253 human patient tissue samples including normal and cancerous tissues from breast, lung, thyroid, and ovary. The mass spectra obtained presented rich molecular profiles characterized by a variety of potential cancer biomarkers identified as metabolites, lipids, and proteins. Statistical classifiers built from the histologically validated molecular database allowed cancer prediction with high sensitivity (96.4%), specificity (96.2%), and overall accuracy (96.3%), as well as prediction of benign and malignant thyroid tumors and different histologic subtypes of lung cancer. Notably, our classifier allowed accurate diagnosis of cancer in marginal tumor regions presenting mixed histologic composition. Last, we demonstrate that the MasSpec Pen is suited for in vivo cancer diagnosis during surgery performed in tumor-bearing mouse models, without causing any observable tissue harm or stress to the animal. Our results provide evidence that the MasSpec Pen could potentially be used as a clinical and intraoperative technology for ex vivo and in vivo cancer diagnosis. PMID:28878011

In Vitro, Ex Vivo and In Vivo Techniques to Study Neuronal Migration in the Developing Cerebral Cortex

PubMed Central

Azzarelli, Roberta; Oleari, Roberto; Lettieri, Antonella; Andre', Valentina; Cariboni, Anna

2017-01-01

Neuronal migration is a fundamental biological process that underlies proper brain development and neuronal circuit formation. In the developing cerebral cortex, distinct neuronal populations, producing excitatory, inhibitory and modulatory neurotransmitters, are generated in different germinative areas and migrate along various routes to reach their final positions within the cortex. Different technical approaches and experimental models have been adopted to study the mechanisms regulating neuronal migration in the cortex. In this review, we will discuss the most common in vitro, ex vivo and in vivo techniques to visualize and study cortical neuronal migration. PMID:28448448

The misleading nature of in vitro and ex vivo findings in studying the impact of stress hormones on NK cell cytotoxicity.

PubMed

Gotlieb, Neta; Rosenne, Ella; Matzner, Pini; Shaashua, Lee; Sorski, Liat; Ben-Eliyahu, Shamgar

2015-03-01

In vitro and ex vivo studies assessing the impact of stress hormones on immune competence commonly replace the natural milieu of leukocytes with an artificial medium, excluding plasma factors, hormones, and cytokines. Given prevalent inconsistencies between in vitro, ex vivo, and in vivo findings, we studied whether such procedures could yield misleading outcomes regarding the impact of stress hormones on NK cell cytotoxicity (NKCC), using fresh human whole blood samples. We found that in the presence of plasma 10-30-fold higher concentrations of cortisol, epinephrine, and prostaglandin-E2 (PGE2) were required to reach suppression levels evident in the context of artificial medium. Importantly, whereas the NK suppressive effects of PGE2 occurred immediately and remained stable upon prolonged exposure, the suppressive effects of cortisol slowly increased over time. Last, to simulate the exclusion of stress factors in the ex vivo approach, we subjected whole blood to stress hormones (as occurs in vivo), and abruptly removed them. We found that the effects of epinephrine and PGE2 quickly disappeared, while the effects of cortisol persisted. Overall, these findings demonstrate the potential misleading nature of in vitro and ex vivo procedures, and specifically suggest that (i) the common in vitro findings of profound suppression of NKCC by stress hormones are overestimation of their direct effects expected in vivo; and (ii) the common ex vivo approach cannot reflect the direct in vivo suppressive effects of epinephrine and PGE2 on NKCC, while inflating the effects of glucocorticoids. Some of these fallacies may be circumvented by using non-delayed whole blood NKCC assays in humans. Copyright © 2014 Elsevier Inc. All rights reserved.

Improving ex vivo skin permeation of non-steroidal anti-inflammatory drugs: enhancing extemporaneous transformation of liposomes into planar lipid bilayers.

PubMed

Vázquez-González, Martha L; Bernad, Rafael; Calpena, Ana C; Domènech, Oscar; Montero, M T; Hernández-Borrell, Jordi

2014-01-30

Transdermal delivery of active principles is a versatile method widely used in medicine. The main drawback for the transdermal route, however, is the low efficiency achieved in the absorption of many drugs, mostly due to the complexity of the skin barrier. To improve drug delivery through the skin, we prepared and characterized liposomes loaded with ibuprofen and designed pharmaceutical formulations based on the extemporaneous addition of penetration enhancer (PE) surfactants. Afterwards, permeation and release studies were carried out. According to the permeation studies, the ibuprofen liposomal formulation supplemented with PEs exhibited similar therapeutic effects, but at lower doses (20%) comparing with a commercial formulation used as a reference. Atomic force microscopy (AFM) was used to investigate the effect caused by PEs on the adsorption mechanism of liposomal formulations onto the skin. Non-fused liposomes, bilayers and multilayered lipid structures were observed. The transformation of vesicles into planar structures is proposed as a possible rationale for explaining the lower doses required when a liposome formulation is supplemented with surfactant PEs. Copyright © 2013 Elsevier B.V. All rights reserved.

In- and ex-vivo molecular imaging of apoptosis to assess sensitivity of non-small cell lung cancer to EGFR inhibitors using probe-based confocal laser endomicroscopy.

PubMed

Guisier, Florian; Bohn, Pierre; Patout, Maxime; Piton, Nicolas; Farah, Insaf; Vera, Pierre; Thiberville, Luc; Salaün, Mathieu

2017-01-01

Prediction of treatment outcome of non-small cell lung cancer (NSCLC) with EGFR inhibitors on the basis of the genetic analysis of the tumor can be incorrect in case of rare or complex mutations, bypass molecular activation pathways, or pharmacodynamic variations. The aim of this study was to develop an ex vivo and in vivo real-time quantitative imaging test for EGFR inhibitors sensitivity assessment. Erlotinib resistant (A549, H460, H1975), insensitive (H1650) and hypersensitive (HCC827) cell lines were injected subcutaneously in Nude mice. Tumor xenografts from mice treated with Erlotinib were imaged ex vivo and in vivo using probe-based confocal laser endomicroscopy (pCLE) and NucView 488 Caspase 3 substrate, a fluorescent probe specific for the activated caspase 3. Assessment of apoptosis at 24h post treatment, both ex vivo in explanted tumor xenografts and in vivo, showed a significant difference between resistant cell lines (A549, H460 and H1975) and insensitive (H1650) or hypersensitive (HCC827) ones (p<0.05 for ex vivo imaging, p≤0.02 for in vivo imaging). There was also a significant difference between insensitive and hypersensitive cell lines, both ex vivo (p<0.05) and in vivo (p = 0.01). Real-time in vivo and ex vivo assessment of apoptosis using pCLE differentiates resistant from sensitive NSCLC xenografts to Erlotinib.

Pharmacological preconditioning for short-term ex vivo expansion of human umbilical cord blood hematopoietic stem cells by filgrastim

PubMed Central

Grigoriadis, Nikolaos G; Grigoriadis, Ioannis G; Markoula, Sofia; Paschopoulos, Minas; Zikopoulos, Konstantinos; Apostolakopoulos, Panagiotis Gr; Vizirianakis, Ioannis S; Georgiou, Ioannis

2016-01-01

Although umbilical cord blood (UCB) hematopoietic stem cell transplantation (UCBT) has emerged as a promising haematological reconstitution therapy for leukemias and other related disorders, the insufficient UCB stem cell dosage still hinders better clinical outcomes. Previous research efforts, by focusing on ex vivo UCB expansion capabilities have sought to benefit from well-known mechanisms of self-renewal characteristics of UCB stem cells. However, the long-term (> 21 days) in vitro culture period and the low neutrophil recovery significantly reduce the transplantability of such ex vivo expanded UCB stem cells. To overcome the latter hurdles in this study, a post-thaw, short-term ex vivo expansion methodology of UCB mononuclear (UCB-MN) and CD34+ cells has been established. Notably, such effort was achieved through pharmacological preconditioned of UCB cultures by filgrastim agent already used in the clinical setting. In crucial cell populations implicated in the promotion of functional engraftment, the progression of free survival rates (PFS), a marked increase of 6.65 to 9.34 fold for UCB-MN and 35 to 49 fold for CD34+ cells has been noticed. Overall, these results indicate that transplantation of pharmacologically-preconditioned ex vivo expansion of UCB stem and progenitor cells keep high promise upon transplantation to enhance therapeutic potential in everyday clinical practice. PMID:27335700

21 CFR 520.246 - Butorphanol tartrate tablets.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Butorphanol tartrate tablets. 520.246 Section 520.246 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... tartrate tablets. (a) Specifications. Each tablet contains 1, 5, or 10 milligrams of butorphanol base...

In vitro and ex vivo angiogenic effects of roxarsone on rat endothelial cells.

PubMed

Zhu, Jiaqiao; Cui, Weibo; Liu, Xue; Ying, Jun; Hu, Chengyun; Zhang, Yumei

2013-11-25

Roxarsone, a feed additive, is being used worldwide to promote animal growth. However, the potential effect of roxarsone on angiogenesis has not been extensively characterized. We examined the ability of roxarsone to promote angiogenesis of rat endothelial cells in vitro and from rat aorta rings ex vivo. Endothelial cells from rats were exposed to 0.01-10.00μM roxarsone, 5ng/mL vascular endothelial growth factor (VEGF) as a positive control or phosphate buffer saline (PBS) as a negative control. Cell proliferation was measured by MTT assay, and the content of VEGF in supernatants was measured by enzyme-linked immunosorbent assay and Western blotting. A Matrigel-induced tube formation assay was used to evaluate the effects of roxarsone on endothelial cells. Additionally, the total number and length of microvessels sprouted from rat aortic rings were measured for ex vivo investigation of angiogenesis. Results showed that the cell viability and total number and length of capillary-like tube formations after roxarsone treatment was significantly higher than that of negative (P<0.05), with a maximum effect at 1.00μM exposure. Furthermore, the number of microvessels sprouted from aortic rings treated for 4h with 0.1-10.0μM roxarsone was significantly higher than that of PBS treatment, with a peak value of 1.0μM. These results further demonstrate the potential of roxarsone to promote angiogenesis in vitro and ex vivo. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Superior survival of ex vivo cultured human reticulocytes following transfusion into mice.

PubMed

Kupzig, Sabine; Parsons, Stephen F; Curnow, Elinor; Anstee, David J; Blair, Allison

2017-03-01

The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34 + cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>10 5 -fold) was achieved using CD34 + cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required. Copyright© Ferrata Storti Foundation.

Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats.

PubMed

Gadi, Dounia; Bnouham, Mohamed; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Legrand, Chantal; Lafeve, Françoise Fauvel; Mekhfi, Hassane

2009-08-17

Many cardiovascular diseases are associated with an increase in blood platelet activity. In Morocco, parsley (Petroselinum crispum, Apiaceae) is one of the medicinal herbs used to treat cardiovascular diseases such as arterial hypertension. In this study, crude aqueous extract (CAE) of parsley was evaluated for its anti-platelet activity in experimental animals on platelet aggregation in vitro and ex vivo; and on bleeding time in vivo. The in vitro aggregation was monitored after pre-incubation of platelets with CAE. The bleeding time and ex vivo aggregation were performed after oral treatment. CAE inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine. The oral administration of CAE (3g/kg) inhibited significantly (p<0.001) platelet aggregation ex vivo and prolonged bleeding time (p<0.001) without changes in the platelet amount. The prolongation of bleeding time by CAE may be attributed to the observed inhibition of platelet aggregation. These effects could be related in part to the polyphenolic compounds present in the extract. These results support the hypothesis that the dietary intake of parsley may be benefit in the normalization of platelet hyperactivation, in the nutritional prevention of cardiovascular diseases and are potentially interesting in the development of new prevention strategies.

Effect of laser generated shockwaves 1 on ex-vivo pigskin.

PubMed

Ramaprasad, Vidyunmala; Navarro, Artemio; Patel, Shahzad; Patel, Vikash; Nowroozi, Bryan N; Taylor, Zach D; Yong, William; Gupta, Vijay; Grundfest, Warren S

2014-10-01

Persistent bacterial infection prolongs hospitalizations, leading to increased healthcare costs. Treatment of these infections costs several billion dollars annually. Biofilm production is one mechanism by which bacteria become resistant. With the help of biofilms, bacteria withstand the host immune response and are much less susceptible to antibiotics. Currently, there is interest in the use of laser-generated shockwaves (LGS) to delaminate biofilm from infected wound surfaces; however, the safety of such an approach has not yet been established. Of particular concern are the thermal and mechanical effects of the shockwave treatment on the epidermis and the underlying collagen structure of the dermis. The present study is a preliminary investigation of the effect of LGS on freshly harvested ex vivo porcine skin tissue samples. Tissue samples for investigation were harvested immediately post-mortem and treated with LGS within 30 minutes. Previous studies have shown that laser fluences between 100 and 500 mJ/pulse are capable of delaminating biofilms off a variety of surfaces, thus our preliminary investigation focused on this range of laser energy. For each sample, LGS were produced via laser irradiation of a thin layer (0.5 µm) of titanium sandwiched between a 50 and 100 µm thick layer of water glass and a 0.1 mm thick sheet of Mylar. The rapid thermal expansion of the irradiated titanium film generates a transient compressive wave that is coupled through a liquid layer to the surface of the ex vivo pigskin sample. Shocked samples were immediately fixed in formalin and prepared for histological analysis. A blinded pathologist evaluated and scored each section on the basis of its overall appearance (O) and presence of linear/slit-like spaces roughly parallel to the surface of the skin (S). The scores were given on a scale of 0-3. The present investigation revealed no visible difference between the tissue sections of the control sample and those that

Formulation, characterization, and in vitro/ex vivo evaluation of quercetin-loaded microemulsion for topical application.

PubMed

Kajbafvala, Azar; Salabat, Alireza; Salimi, Anayatollah

2016-12-09

The aim of this study was to develop a new microemulsion formulation for topical application of poorly soluble drug named quercetin. In order to design suitable microemulsion system, the pseudo-ternary phase diagrams of microemulsion systems were constructed at different surfactant/co-surfactant ratios using tween 80 as surfactant, transcutol ® P as a co-surfactant and oleic acid as an oil phase. Some physicochemical properties such as droplet size, density, refractive index, electrical conductivity, pH, surface tension, and viscosity of the microemulsion systems were measured at 298.15 K. The average hydrodynamic droplet size of the optimized microemulsions was obtained by dynamic light scattering method. Morphology assessment of the optimized quercetin-loaded microemulsion by transmission electron microscopy analysis indicated that the particles have the size of about 25 nm and spherical with narrow size distribution. Equilibrium solubility, in vitro drug release at a 24 h time period, release kinetic evaluation as well as ex vivo permeation and retention of quercetin-loaded microemulsions through rat skin has been investigated. The obtained results showed a slow release behavior without any transdermal delivery. Most of the formulations fitted best with zero-order kinetic model with a non-Fickian mechanisms. This study illustrated that the proposed QU-microemulsion has a good potential for use in sunscreen formulations. [Formula: see text].

Micron-sized and submicron-sized aerosol deposition in a new ex vivo preclinical model.

PubMed

Perinel, Sophie; Leclerc, Lara; Prévôt, Nathalie; Deville, Agathe; Cottier, Michèle; Durand, Marc; Vergnon, Jean-Michel; Pourchez, Jérémie

2016-07-07

The knowledge of where particles deposit in the respiratory tract is crucial for understanding the health effects associated with inhaled drug particles. An ex vivo study was conducted to assess regional deposition patterns (thoracic vs. extrathoracic) of radioactive polydisperse aerosols with different size ranges [0.15 μm-0.5 μm], [0.25 μm-1 μm] and [1 μm-9 μm]. SPECT/CT analyses were performed complementary in order to assess more precisely the regional deposition of aerosols within the pulmonary tract. Experiments were set using an original respiratory tract model composed of a human plastinated head connected to an ex vivo porcine pulmonary tract. The model was ventilated by passive expansion, simulating pleural depressions. Aerosol was administered during nasal breathing. Planar scintigraphies allowed to calculate the deposited aerosol fractions for particles in the three size ranges from sub-micron to micron The deposited fractions obtained, for thoracic vs. extra-thoracic regions respectively, were 89 ± 4 % vs. 11 ± 4 % for [0.15 μm-0.5 μm], 78 ± 5 % vs. 22 ± 5 % for [0.25 μm-1 μm] and 35 ± 11 % vs.65 ± 11 % for [1 μm-9 μm]. Results obtained with this new ex vivo respiratory tract model are in good agreement with the in vivo data obtained in studies with baboons and humans.

Spectroscopic optical coherence tomography for ex vivo brain tumor analysis

NASA Astrophysics Data System (ADS)

Lenz, Marcel; Krug, Robin; Dillmann, Christopher; Gerling, Alexandra; Gerhardt, Nils C.; Welp, Hubert; Schmieder, Kirsten; Hofmann, Martin R.

2017-02-01

For neurosurgeries precise tumor resection is essential for the subsequent recovery of the patients since nearby healthy tissue that may be harmed has a huge impact on the life quality after the surgery. However, so far no satisfying methodology has been established to assist the surgeon during surgery to distinguish between healthy and tumor tissue. Optical Coherence Tomography (OCT) potentially enables non-contact in vivo image acquisition at penetration depths of 1-2 mm with a resolution of approximately 1-15 μm. To analyze the potential of OCT for distinction between brain tumors and healthy tissue, we used a commercially available Thorlabs Callisto system to measure healthy tissue and meningioma samples ex vivo. All samples were measured with the OCT system and three dimensional datasets were generated. Afterwards they were sent to the pathology for staining with hematoxylin and eosin and then investigated with a bright field microscope to verify the tissue type. This is the actual gold standard for ex vivo analysis. The images taken by the OCT system exhibit variations in the structure for different tissue types, but these variations may not be objectively evaluated from raw OCT images. Since an automated distinction between tumor and healthy tissue would be highly desirable to guide the surgeon, we applied Spectroscopic Optical Coherence Tomography to further enhance the differences between the tissue types. Pattern recognition and machine learning algorithms were applied to classify the derived spectroscopic information. Finally, the classification results are analyzed in comparison to the histological analysis of the samples.

High resolution SAW elastography for ex-vivo porcine skin specimen

NASA Astrophysics Data System (ADS)

Zhou, Kanheng; Feng, Kairui; Wang, Mingkai; Jamera, Tanatswa; Li, Chunhui; Huang, Zhihong

2018-02-01

Surface acoustic wave (SAW) elastography has been proven to be a non-invasive, non-destructive method for accurately characterizing tissue elastic properties. Current SAW elastography technique tracks generated surface acoustic wave impulse point by point which are a few millimeters away. Thus, reconstructed elastography has low lateral resolution. To improve the lateral resolution of current SAW elastography, a new method was proposed in this research. A M-B scan mode, high spatial resolution phase sensitive optical coherence tomography (PhS-OCT) system was employed to track the ultrasonically induced SAW impulse. Ex-vivo porcine skin specimen was tested using this proposed method. A 2D fast Fourier transform based algorithm was applied to process the acquired data for estimating the surface acoustic wave dispersion curve and its corresponding penetration depth. Then, the ex-vivo porcine skin elastogram was established by relating the surface acoustic wave dispersion curve and its corresponding penetration depth. The result from the proposed method shows higher lateral resolution than that from current SAW elastography technique, and the approximated skin elastogram could also distinguish the different layers in the skin specimen, i.e. epidermis, dermis and fat layer. This proposed SAW elastography technique may have a large potential to be widely applied in clinical use for skin disease diagnosis and treatment monitoring.

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by In Vivo Whole Animal and Ex Vivo Super-Resolution Fluorescence Imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chien, Miao-Ping; Carlini, Andrea S.; Hu, Dehong

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block, and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo, and in ex vivo organ analysis following intratumoral injectionmore » into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micron-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.« less

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study.

PubMed

Candiota, Ana Paula; Acosta, Milena; Simões, Rui Vasco; Delgado-Goñi, Teresa; Lope-Piedrafita, Silvia; Irure, Ainhoa; Marradi, Marco; Bomati-Miguel, Oscar; Miguel-Sancho, Nuria; Abasolo, Ibane; Schwartz, Simó; Santamaria, Jesús; Penadés, Soledad; Arús, Carles

2014-04-05

Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study

PubMed Central

2014-01-01

Background Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. Results The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. Conclusion We have developed a simple, cost-effective relatively high-throughput method for

Rapid ex vivo imaging of PAIII prostate to bone tumor with SWIFT-MRI.

PubMed

Luhach, Ihor; Idiyatullin, Djaudat; Lynch, Conor C; Corum, Curt; Martinez, Gary V; Garwood, Michael; Gillies, Robert J

2014-09-01

The limiting factor for MRI of skeletal/mineralized tissue is fast transverse relaxation. A recent advancement in MRI technology, SWIFT (Sweep Imaging with Fourier Transform), is emerging as a new approach to overcome this difficulty. Among other techniques like UTE, ZTE, and WASPI, the application of SWIFT technology has the strong potential to impact preclinical and clinical imaging, particularly in the context of primary or metastatic bone cancers because it has the added advantage of imaging water in mineralized tissues of bone allowing MRI images to be obtained of tissues previously visible only with modalities such as computed tomography (CT). The goal of the current study is to examine the feasibility of SWIFT for the assessment of the prostate cancer induced changes in bone formation (osteogenesis) and destruction (osteolysis) in ex vivo specimens. A luciferase expressing prostate cancer cell line (PAIII) or saline control was inoculated directly into the tibia of 6-week-old immunocompromised male mice. Tumor growth was assessed weekly for 3 weeks before euthanasia and dissection of the tumor bearing and sham tibias. The ex vivo mouse tibia specimens were imaged with a 9.4 Tesla (T) and 7T MRI systems. SWIFT images are compared with traditional gradient-echo and spin-echo MRI images as well as CT and histological sections. SWIFT images with nominal resolution of 78 μm are obtained with the tumor and different bone structures identified. Prostate cancer induced changes in the bone microstructure are visible in SWIFT images, which is supported by spin-echo, high resolution CT and histological analysis. SWIFT MRI is capable of high-quality high-resolution ex vivo imaging of bone tumor and surrounding bone and soft tissues. Furthermore, SWIFT MRI shows promise for in vivo bone tumor imaging, with the added benefits of nonexposure to ionizing radiation, quietness, and speed. Copyright © 2013 Wiley Periodicals, Inc.

Imaging denatured collagen strands in vivo and ex vivo via photo-triggered hybridization of caged collagen mimetic peptides.

PubMed

Li, Yang; Foss, Catherine A; Pomper, Martin G; Yu, S Michael

2014-01-31

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Experimental analysis of insertion torques and forces of threaded and press-fit acetabular cups by means of ex vivo and in vivo measurements.

PubMed

Vogel, Danny; Rathay, Andreas; Teufel, Stephanie; Ellenrieder, Martin; Zietz, Carmen; Sander, Manuela; Bader, Rainer

2017-01-01

In THA a sufficient primary implant stability is the precondition for successful secondary stability. Industrial foams of different densities have been used for primary stability investigations. The aim of this study was to analyse and compare the insertion behaviour of threaded and press-fit cups in vivo and ex vivo using bone substitutes with various densities. Two threaded (Bicon Plus®, Trident® TC) and one press-fit cup (Trident PSL®) were inserted by orthopaedic surgeons (S1, S2) into 10, 20 and 31 pcf blocks, using modified surgical instruments allowing measurements of the insertion forces and torques. Furthermore, the insertion behaviour of two cups were analysed intraoperatively. Torques for the threaded cups increased while bone substitute density increased. Maximum insertion torques were observed for S2 with 102 Nm for the Bicon Plus® in 20 pcf blocks and 77 Nm for the Trident® TC in 31 pcf blocks, which compares to the in vivo measurement (85 Nm). The average insertion forces for the press-fit cup varied from 5.2 to 6.8 kN (S1) and 7.2-11.5 kN (S2) ex vivo. Intraoperatively an average insertion force of 8.0 kN was determined. Implantation behaviour was influenced by acetabular cup design, bone substitute and experience of the surgeon. No specific density of bone substitute could be favoured for ex vivo investigations on the implantation behaviour of acetabular cups. The use synthetic bone blocks of high density (31 pcf) led to problems regarding cup orientation and seating. Therefore, bone substitutes used should be critically scrutinized in terms of the comparability to the in vivo situation.

Clinical indices of in vivo biocompatibility: the role of ex vivo cell function studies and effluent markers in peritoneal dialysis patients.

PubMed

Mackenzie, Ruth; Holmes, Clifford J; Jones, Suzanne; Williams, John D; Topley, Nicholas

2003-12-01

Clinical indices of in vivo biocompatibility: The role of ex vivo cell function studies and effluent markers in peritoneal dialysis patients. Over the past 20 years, studies of the biocompatibility profile of peritoneal dialysis solutions (PDF) have evolved from initial in vitro studies assessing the impact of solutions on leukocyte function to evaluations of mesothelial cell behavior. More recent biocompatibility evaluations have involved assessments of the impact of PDF on membrane integrity and cell function in peritoneal dialysis (PD) patients. The development of ex vivo systems for the evaluation of in vivo cell function, and effluent markers of membrane integrity and inflammation in patients exposed both acutely and chronically to conventional and new PDF will be interpreted in the context of our current understanding of the biology of the dialyzed peritoneum. The available data indicate that exposure of the peritoneal environment to more biocompatible PDF is associated with improvements in peritoneal cell function, alterations in markers of membrane integrity, and reduced local inflammation. These data suggest that more biocompatible PDF will have a positive impact on host defense, peritoneal homeostasis, and the long-term preservation of peritoneal membrane function in PD patients.

An ex vivo approach to botanical-drug interactions: a proof of concept study.

PubMed

Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J; Markowitz, John S

2015-04-02

Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodology in applied ethnopharmacological research. This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal׳s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybin A, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more

An ex vivo approach to botanical-drug interactions: A proof of concept study

PubMed Central

Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J.; Markowitz, John S.

2015-01-01

Ethnopharmacological relevance Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodoloy in applied ethnopharmacological research. Materials and Methods This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Results Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal’s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybinA, silybin B and hydrastine and berberine

A protocol to study ex vivo mouse working heart at human-like heart rate.

PubMed

Feng, Han-Zhong; Jin, Jian-Ping

2018-01-01

Genetically modified mice are widely used as experimental models to study human heart function and diseases. However, the fast rate of normal mouse heart at 400-600bpm limits its capacity of assessing kinetic parameters that are important for the physiology and pathophysiology of human heart that beats at a much slower rate (75-180bpm). To extend the value of mouse models, we established a protocol to study ex vivo mouse working hearts at a human-like heart rate. In the presence of 300μM lidocaine to lower pacemaker and conductive activities and prevent arrhythmia, a stable rate of 120-130bpm at 37°C is achieved for ex vivo mouse working hearts. The negative effects of decreased heart rate on force-frequency dependence and lidocaine as a myocardial depressant on intracellular calcium can be compensated by using a higher but still physiological level of calcium (2.75mM) in the perfusion media. Multiple parameters were studied to compare the function at the human-like heart rate with that of ex vivo mouse working hearts at the standard rate of 480bpm. The results showed that the conditions for slower heart rate in the presence of 300μM lidocaine did not have depressing effect on left ventricular pressure development, systolic and diastolic velocities and stroke volume with maintained positive inotropic and lusitropic responses to β-adrenergic stimulation. Compared with that at 480bpm, the human-like heart rate increased ventricular filling and end diastolic volume with enhanced Frank-Starling responses. Coronary perfusion was increased from longer relaxation time and interval between beats whereas cardiac efficiency was significantly improved. Although the intrinsic differences between mouse and human heart remain, this methodology for ex vivo mouse hearts to work at human-like heart rate extends the value of using genetically modified mouse models to study cardiac function and human heart diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Preparation, Optimization, In vitro Evaluation and Ex vivo Permeation Studies of Finasteride loaded gel Formulations Prepared by using Response Surface Methodology.

PubMed

Khan, Muhammad Zia Ullah; Makreski, Petre; Murtaza, Ghulam

2018-05-02

The aim of present explorative study was to prepare and optimize finasteride loaded topical gel formulations by using three factor [propylene glycol (PG), Tween® 80, and sodium lauryl sulphate (SLS)], five level central composite design. Optimized finasteride topical gel formulation (F4), containing PG, Tween® 80, and SLS in a concentration of 0.8 mg, 0.4 mg and 0.2 mg, respectively, showed 6-fold higher values of cumulative drug release, flux, partition coefficient, input rate, lag time, and diffusion coefficient, when compared to control formulation without permeation enhancer. Finally, it can be concluded that finasteride permeation was enhanced by PG, tween® 80 and SLS individually, while in combination only PG along with tween® 80 had synergistic and more pronounced effect on flux, permeability coefficient and input rate while antagonistic effect on lag time and diffusion coefficient was observed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A randomized comparison of laparoscopic, flexible endoscopic, and wired and wireless magnetic cameras on ex vivo and in vivo NOTES surgical performance.

PubMed

Chang, Victoria C; Tang, Shou-Jiang; Swain, C Paul; Bergs, Richard; Paramo, Juan; Hogg, Deborah C; Fernandez, Raul; Cadeddu, Jeffrey A; Scott, Daniel J

2013-08-01

The influence of endoscopic video camera (VC) image quality on surgical performance has not been studied. Flexible endoscopes are used as substitutes for laparoscopes in natural orifice translumenal endoscopic surgery (NOTES), but their optics are originally designed for intralumenal use. Manipulable wired or wireless independent VCs might offer advantages for NOTES but are still under development. To measure the optical characteristics of 4 VC systems and to compare their impact on the performance of surgical suturing tasks. VC systems included a laparoscope (Storz 10 mm), a flexible endoscope (Olympus GIF 160), and 2 prototype deployable cameras (magnetic anchoring and guidance system [MAGS] Camera and PillCam). In a randomized fashion, the 4 systems were evaluated regarding standardized optical characteristics and surgical manipulations of previously validated ex vivo (fundamentals of laparoscopic surgery model) and in vivo (live porcine Nissen model) tasks; objective metrics (time and errors/precision) and combined surgeon (n = 2) performance were recorded. Subtle differences were detected for color tests, and field of view was variable (65°-115°). Suitable resolution was detected up to 10 cm for the laparoscope and MAGS camera but only at closer distances for the endoscope and PillCam. Compared with the laparoscope, surgical suturing performances were modestly lower for the MAGS camera and significantly lower for the endoscope (ex vivo) and PillCam (ex vivo and in vivo). This study documented distinct differences in VC systems that may be used for NOTES in terms of both optical characteristics and surgical performance. Additional work is warranted to optimize cameras for NOTES. Deployable systems may be especially well suited for this purpose.

Ex-vivo expansion of red blood cells: How real for transfusion in humans?

PubMed Central

Migliaccio, Anna Rita; Masselli, Elena; Varricchio, Lilian; Whitsett, Carolyn

2013-01-01

Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5 × 1012 cells vs 107 cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality. PMID:22177597

Human Skin Permeation Studies with PPARγ Agonist to Improve Its Permeability and Efficacy in Inflammatory Processes.

PubMed

Silva-Abreu, Marcelle; Espinoza, Lupe Carolina; Rodríguez-Lagunas, María José; Fábrega, María-José; Espina, Marta; García, María Luisa; Calpena, Ana Cristina

2017-11-28

Rosacea is the most common inflammatory skin disease. It is characterized by erythema, inflammatory papules and pustules, visible blood vessels, and telangiectasia. The current treatment has limitations and unsatisfactory results. Pioglitazone (PGZ) is an agonist of peroxisome proliferator-activated receptors (PPARs), a nuclear receptor that regulates important cellular functions, including inflammatory responses. The purpose of this study was to evaluate the permeation of PGZ with a selection of penetration enhancers and to analyze its effectiveness for treating rosacea. The high-performance liquid chromatography (HPLC) method was validated for the quantitative determination of PGZ. Ex vivo permeation experiments were realized in Franz diffusion cells using human skin, in which PGZ with different penetration enhancers were assayed. The results showed that the limonene was the most effective penetration enhancer that promotes the permeation of PGZ through the skin. The cytotoxicity studies and the Draize test detected cell viability and the absence of skin irritation, respectively. The determination of the skin color using a skin colorimetric probe and the results of histopathological studies confirmed the ability of PGZ-limonene to reduce erythema and vasodilation. This study suggests new pharmacological indications of PGZ and its possible application in the treatment of skin diseases, namely rosacea.

Transgene delivery to endothelial cultures derived from porcine carotid artery ex vivo.

PubMed

Andoh, J; Sawyer, B; Szewczyk, K; Nortley, M; Rossetti, T; Loftus, I M; Yáñez-Muñoz, R J; Hainsworth, A H

2013-10-01

Carotid artery disease is a widespread cause of morbidity and mortality. Porcine models of vascular disease are well established in vivo, but existing endothelial systems in vitro (e.g. human umbilical vein endothelial cells, rat aortic endothelial cultures) poorly reflect carotid endothelium. A reliable in vitro assay would improve design of in vivo experiments and allow reduction and refinement of animal use. This study aimed (1) to develop ex vivo endothelial cultures from porcine carotid and (2) to test whether these were suitable for lentivector-mediated transgene delivery. Surplus carotid arteries were harvested from young adult female Large White pigs within 10 min post-mortem. Small sectors of carotid artery wall (approximately 4 mm×4 mm squares) were immobilised in a stable gel matrix. Cultures were exposed to HIV-derived lentivector (LV) encoding a reporter transgene or the equivalent integration-deficient vector (IDLV). After 7-14 days in vitro, cultures were fixed and labelled histochemically. Thread-like multicellular outgrowths were observed that were positive for endothelial cell markers (CD31, VEGFR2, von Willebrand factor). A minority of cells co-labelled for smooth muscle markers. Sensitivity to cytotoxic agents (paclitaxel, cycloheximide, staurosporine) was comparable to that in cell cultures, indicating that the gel matrix permits diffusive access of small pharmacological molecules. Transgene-expressing cells were more abundant following exposure to LV than IDLV (4.7, 0.1% of cells, respectively). In conclusion, ex vivo adult porcine carotid artery produced endothelial cell outgrowths that were effectively transduced by LV. This system will facilitate translation of novel therapies to clinical trials, with reduction and refinement of in vivo experiments.

Helicobacter pylori-induced gastric pathology: insights from in vivo and ex vivo models

PubMed Central

Williams, Jonathan M.

2017-01-01

ABSTRACT Gastric colonization with Helicobacter pylori induces diverse human pathological conditions, including superficial gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma and its precursors. The treatment of these conditions often relies on the eradication of H. pylori, an intervention that is increasingly difficult to achieve and that does not prevent disease progression in some contexts. There is, therefore, a pressing need to develop new experimental models of H. pylori-associated gastric pathology to support novel drug development in this field. Here, we review the current status of in vivo and ex vivo models of gastric H. pylori colonization, and of Helicobacter-induced gastric pathology, focusing on models of gastric pathology induced by H. pylori, Helicobacter felis and Helicobacter suis in rodents and large animals. We also discuss the more recent development of gastric organoid cultures from murine and human gastric tissue, as well as from human pluripotent stem cells, and the outcomes of H. pylori infection in these systems. PMID:28151409

Chest drainage teaching and training for medical students. Use of a surgical ex vivo pig model.

PubMed

Tube, Milton Ignacio Carvalho; Netto, Fernando Antonio Campelo Spencer; Costa, Elaine; Lafayette, Daniell de Siqueira Araújo; Lima, George Augusto da Fonseca Carvalho Antunes; Menezes, Jamile Isabela Santos de; Aires, Vinicius Gueiros Buenos; Ferraz, Álvaro Antônio Bandeira; Campos, Josemberg Marins; Moraes, Fernando Ribeiro de

2016-05-01

Implement a constructivist approach in thoracic drainage training in surgical ex vivo pig models, to compare the acquisition of homogeneous surgical skills between medical students. Experimental study, prospective, transversal, analytical, controlled, three steps. Selection, training, evaluation. a) students without training in thoracic drainage; b) without exposure to constructivist methodology. 2) EXCLUSION CRITERIA: a) students developed surgical skills; b) a history of allergy. (N = 312). Two groups participated in the study: A and B. Lecture equal for both groups. Differentiated teaching: group A, descriptive and informative method; group B, learning method based on problems. A surgical ex vivo pig model for training the chest drain was created. Were applied pre and post-test, test goal-discursive and OSATS scale. Theoretical averages: Group A = 9.5 ± 0.5; Group B = 8.8 ± 1.1 (p = 0.006). Medium Practices: Group A = 22.8 ± 1.8; Group B = 23.0 ± 2.8 (p <0.001). Through the constructivist methodology implemented in the thoracic drainage training in surgical ex vivo pig models, has proven the acquisition of surgical skills homogeneous compared among medical students.

First identification of the herpes simplex virus by skin-dedicated ex vivo fluorescence confocal microscopy during herpetic skin infections.

PubMed

Cinotti, E; Perrot, J L; Labeille, B; Campolmi, N; Thuret, G; Naigeon, N; Bourlet, T; Pillet, S; Cambazard, F

2015-06-01

Skin-dedicated ex vivo fluorescence confocal microscopy (FCM) has so far been used to identify cutaneous tumours on freshly excised samples using acridine orange as fluorochrome. To use FCM for a new indication, namely, the identification of the herpes simplex virus (HSV) in skin lesions, using fluorescent antibodies. Six roof samples from skin vesicles suspicious for HSV lesions were incubated with anti-HSV-1 and anti-HSV-2 antibodies coupled with fluorescein isothiocyanate, and examined under skin-dedicated ex vivo FCM. The positive controls were swabs taken from the floor of each vesicle and observed under conventional direct fluorescence assay (DFA) and by viral cultures. Roof samples from three bullae of bullous pemphigoid were the negative controls. Using ex vivo FCM, the samples from the lesions clinically suspicious for HSV infection were seen to be fluorescent after incubation with anti-HSV-1, and were negative after incubation with anti-HSV-2 antibodies. Conventional DFA with an optical microscope and cultures confirmed the presence of HSV-1 infection. By using fluorescent antibodies to identify precise structures, ex vivo FCM can be used for indications other than tumour identification. More specifically, it can be an additional diagnostic tool for HSV infection. © 2014 British Association of Dermatologists.

The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.

PubMed

Blanco, Teresa Mortera; Mantalaris, Athanasios; Bismarck, Alexander; Panoskaltsis, Nicki

2010-03-01

Acute myeloid leukaemia (AML) is a cancer of haematopoietic cells that develops in three-dimensional (3-D) bone marrow niches in vivo. The study of AML has been hampered by lack of appropriate ex vivo models that mimic this microenvironment. We hypothesised that fabrication and optimisation of suitable biomimetic scaffolds for culturing leukaemic cells ex vivo might facilitate the study of AML in its native 3-D niche. We evaluated the growth of three leukaemia subtype-specific cell lines, K-562, HL60 and Kasumi-6, on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials, such as poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate), poly (D, L-lactade), poly (caprolactone), and polystyrene. Our results show that PLGA and PU supported the best seeding efficiency and leukaemic growth. Furthermore, the PLGA and PU scaffolds were coated with extracellular matrix (ECM) proteins, collagen type I (62.5 or 125 microg/ml) and fibronectin (25 or 50 microg/ml) to provide biorecognition signals. The 3 leukaemia subtype-specific lines grew best on PU scaffolds coated with 62.5 microg/ml collagen type I over 6 weeks in the absence of exogenous growth factors. In conclusion, PU-collagen scaffolds may provide a practical model to study the biology and treatment of primary AML in an ex vivo mimicry. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

[Ex vivo expansion and clonal variation of CD34(+)CD59(+) cells from bone marrow in children with paroxysmal nocturnal hemoglobinuria].

PubMed

Xiao, Juan; Wu, Yong-Ji; Han, Bing; Dong, Hong-Yan; Chen, Shi-Ping

2013-08-01

To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH. CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted. The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01). The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.

Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model

PubMed Central

Weidensdorfer, Marko; Chae, Ju Ik; Makobe, Celestine; Stahl, Julia; Averhoff, Beate; Müller, Volker; Schürmann, Christoph; Brandes, Ralf P.; Wilharm, Gottfried; Ballhorn, Wibke; Christ, Sara; Linke, Dirk; Fischer, Doris; Göttig, Stephan

2015-01-01

Bacterial adherence determines the virulence of many human-pathogenic bacteria. Experimental approaches elucidating this early infection event in greater detail have been performed using mainly methods of cellular microbiology. However, in vitro infections of cell monolayers reflect the in vivo situation only partially, and animal infection models are not available for many human-pathogenic bacteria. Therefore, ex vivo infection of human organs might represent an attractive method to overcome these limitations. We infected whole human umbilical cords ex vivo with Bartonella henselae or Acinetobacter baumannii under dynamic flow conditions mimicking the in vivo infection situation of human endothelium. For this purpose, methods for quantifying endothelium-adherent wild-type and trimeric autotransporter adhesin (TAA)-deficient bacteria were set up. Data revealed that (i) A. baumannii binds in a TAA-dependent manner to endothelial cells, (ii) this organ infection model led to highly reproducible adherence rates, and furthermore, (iii) this model allowed to dissect the biological function of TAAs in the natural course of human infections. These findings indicate that infection models using ex vivo human tissue samples (“organ microbiology”) might be a valuable tool in analyzing bacterial pathogenicity with the capacity to replace animal infection models at least partially. PMID:26712205

Ex-vivo and live animal models are equally effective training for the management of a penetrating cardiac injury.

PubMed

Izawa, Yoshimitsu; Hishikawa, Shuji; Muronoi, Tomohiro; Yamashita, Keisuke; Maruyama, Hiroyuki; Suzukawa, Masayuki; Lefor, Alan Kawarai

2016-01-01

Live tissue models are considered the most useful simulation for training in the management for hemostasis of penetrating injuries. However, these models are expensive, with limited opportunities for repetitive training. Ex-vivo models using tissue and a fluid pump are less expensive, allow repetitive training and respect ethical principles in animal research. The purpose of this study is to objectively evaluate the effectiveness of ex-vivo training with a pump, compared to live animal model training. Staff surgeons and residents were divided into live tissue training and ex-vivo training groups. Training in the management of a penetrating cardiac injury was conducted for each group, separately. One week later, all participants were formally evaluated in the management of a penetrating cardiac injury in a live animal. There are no differences between the two groups regarding average years of experience or previous trauma surgery experience. All participants achieved hemostasis, with no difference between the two groups in the Global Rating Scale score (ex-vivo: 25.2 ± 6.3, live: 24.7 ± 6.3, p = 0.646), blood loss (1.6 ± 0.7, 2.0 ± 0.6, p = 0.051), checklist score (3.7 ± 0.6, 3.6 ± 0.9, p = 0.189), or time required for repair (101 s ± 31, 107 s ± 15, p = 0.163), except overall evaluation (3.8 ± 0.9, 3.4 ± 0.9, p = 0.037). The internal consistency reliability and inter-rater reliability in the Global Rating Scale were excellent (0.966 and 0.953 / 0.719 and 0.784, respectively), and for the checklist were moderate (0.570 and 0.636 / 0.651 and 0.607, respectively). The validity is rated good for both the Global Rating Scale (Residents: 21.7 ± 5.6, Staff: 28.9 ± 4.7, p = 0.000) and checklist (Residents: 3.4 ± 0.9, Staff Surgeons: 3.9 ± 0.3, p = 0.003). The results of self-assessment questionnaires were similarly high (4.2-4.9) with scores in self-efficacy increased after

Surgical model pig ex vivo for venous dissection teaching in medical schools.

PubMed

Tube, Milton Ignacio Carvalho; Spencer-Netto, Fernando Antonio Campelo; Oliveira, Anderson Igor Pereira de; Holanda, Arthur Cesário de; Barros, Bruno Leão Dos Santos; Rezende, Caio Cezar Gomes; Cavalcanti, João Pedro Guerra; Batista, Marília Apolinário; Campos, Josemberg Marins

2017-02-01

To investigate a method for development of surgical skills in medical students simulating venous dissection in surgical ex vivo pig model. Prospective, analytical, experimental, controlled study with four stages: selection, theoretical teaching, training and assessment. Sample of 312 students was divided into two groups: Group A - 2nd semester students; Group B - students of 8th semester. The groups were divided into five groups of 12 students, trained two hours per week in the semester. They set up four models to three students in each skill station assisted by a monitor. Teaching protocol emergency procedures training were applied to venous dissection, test goal-discursive and OSATS scale. The pre-test confirmed that the methodology has not been previously applied to the students. The averages obtained in the theoretical evaluation reached satisfactory parameters in both groups. The results of applying OSATS scale showed the best performance in group A compared to group B, however, both groups had satisfactory medium. The method was enough to raise a satisfactory level of skill both groups in venous dissection running on surgical swine ex vivo models.

Algorithm optimization for multitined radiofrequency ablation: comparative study in ex vivo and in vivo bovine liver.

PubMed

Appelbaum, Liat; Sosna, Jacob; Pearson, Robert; Perez, Sarah; Nissenbaum, Yizhak; Mertyna, Pawel; Libson, Eugene; Goldberg, S Nahum

2010-02-01

To prospectively optimize multistep algorithms for largest available multitined radiofrequency (RF) electrode system in ex vivo and in vivo tissues, to determine best energy parameters to achieve large predictable target sizes of coagulation, and to compare these algorithms with manufacturer's recommended algorithms. Institutional animal care and use committee approval was obtained for the in vivo portion of this study. Ablation (n = 473) was performed in ex vivo bovine liver; final tine extension was 5-7 cm. Variables in stepped-deployment RF algorithm were interrogated and included initial current ramping to 105 degrees C (1 degrees C/0.5-5.0 sec), the number of sequential tine extensions (2-7 cm), and duration of application (4-12 minutes) for final two to three tine extensions. Optimal parameters to achieve 5-7 cm of coagulation were compared with recommended algorithms. Optimal settings for 5- and 6-cm final tine extensions were confirmed in in vivo perfused bovine liver (n = 14). Multivariate analysis of variance and/or paired t tests were used. Mean RF ablation zones of 5.1 cm +/- 0.2 (standard deviation), 6.3 cm +/- 0.4, and 7 cm +/- 0.3 were achieved with 5-, 6-, and 7-cm final tine extensions in a mean of 19.5 min +/- 0.5, 27.9 min +/- 6, and 37.1 min +/- 2.3, respectively, at optimal settings. With these algorithms, size of ablation at 6- and 7-cm tine extension significantly increased from mean of 5.4 cm +/- 0.4 and 6.1 cm +/- 0.6 (manufacturer's algorithms) (P <.05, both comparisons); two recommended tine extensions were eliminated. In vivo confirmation produced mean diameter in specified time: 5.5 cm +/- 0.4 in 18.5 min +/- 0.5 (5-cm extensions) and 5.7 cm +/- 0.2 in 21.2 min +/- 0.6 (6-cm extensions). Large zones of coagulation of 5-7 cm can be created with optimized RF algorithms that help reduce number of tine extensions compared with manufacturer's recommendations. Such algorithms are likely to facilitate the utility of these devices for RF

Enzyme-directed assembly of nanoparticles in tumors monitored by in vivo whole animal imaging and ex vivo super-resolution fluorescence imaging.

PubMed

Chien, Miao-Ping; Carlini, Andrea S; Hu, Dehong; Barback, Christopher V; Rush, Anthony M; Hall, David J; Orr, Galya; Gianneschi, Nathan C

2013-12-18

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system.

PubMed

McCaughey-Chapman, Amy; Connor, Bronwen

2017-02-01

Organotypic brain slice cultures are a useful tool to study neurological function as they provide a more complex, 3-dimensional system than standard 2-dimensional in vitro cell cultures. Building on a previously developed mouse brain slice culture protocol, we have developed a rat sagittal brain slice culture system as an ex vivo model of dopamine cell loss. We show that rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Using Fluoro-Gold axonal tracing, we demonstrate that the slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period, with particular focus on the nigrostriatal pathway. Treatment of the cultures with 6-hydroxydopamine and desipramine induces a progressive loss of Fluoro-Gold-positive nigral cells with a sustained loss of tyrosine hydroxylase-positive nigral cells. This recapitulates the pattern of dopaminergic degeneration observed in the rat partial 6-hydroxydopamine lesion model and, most importantly, the progressive pathology of Parkinson's disease. Our slice culture platform provides an advance over other systems, as we demonstrate for the first time 3-dimensional cytoarchitecture maintenance of rat nigrostriatal sagittal slices for up to 6 weeks. Our ex vivo organotypic slice culture system provides a long term cellular platform to model Parkinson's disease, allowing for the elucidation of mechanisms involved in dopaminergic neuron degeneration and the capability to study cellular integration and plasticity ex vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

Minimal vascular flows cause strong heat sink effects in hepatic radiofrequency ablation ex vivo.

PubMed

Lehmann, Kai S; Poch, Franz G M; Rieder, Christian; Schenk, Andrea; Stroux, Andrea; Frericks, Bernd B; Gemeinhardt, Ole; Holmer, Christoph; Kreis, Martin E; Ritz, Jörg P; Zurbuchen, Urte

2016-08-01

The present paper aims to assess the lower threshold of vascular flow rate on the heat sink effect in bipolar radiofrequency ablation (RFA) ex vivo. Glass tubes (vessels) of 3.4 mm inner diameter were introduced in parallel to bipolar RFA applicators into porcine liver ex vivo. Vessels were perfused with flow rates of 0 to 1,500 ml/min. RFA (30 W power, 15 kJ energy input) was carried out at room temperature and 37°C. Heat sink effects were assessed in RFA cross sections by the decrease in ablation radius, area and by a high-resolution sector planimetry. Flow rates of 1 ml/min already caused a significant cooling effect (P ≤ 0.001). The heat sink effect reached a maximum at 10 ml/min (18.4 mm/s) and remained stable for flow rates up to 1,500 ml/min. Minimal vascular flows of ≥1 ml/min cause a significant heat sink effect in hepatic RFA ex vivo. A lower limit for volumetric flow rate was not found. The maximum of the heat sink effect was reached at a flow rate of 10 ml/min and remained stable for flow rates up to 1,500 ml/min. Hepatic inflow occlusion should be considered in RFA close to hepatic vessels. © 2016 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

Ex-vivo expansion of red blood cells: how real for transfusion in humans?

PubMed

Migliaccio, Anna Rita; Masselli, Elena; Varricchio, Lilian; Whitsett, Carolyn

2012-03-01

Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5×10(12) cells vs 10(7) cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality. Copyright © 2011 Elsevier Ltd. All rights reserved.

Multiphoton gradient index endoscopy for evaluation of diseased human prostatic tissue ex vivo

NASA Astrophysics Data System (ADS)

Huland, David M.; Jain, Manu; Ouzounov, Dimitre G.; Robinson, Brian D.; Harya, Diana S.; Shevchuk, Maria M.; Singhal, Paras; Xu, Chris; Tewari, Ashutosh K.

2014-11-01

Multiphoton microscopy can instantly visualize cellular details in unstained tissues. Multiphoton probes with clinical potential have been developed. This study evaluates the suitability of multiphoton gradient index (GRIN) endoscopy as a diagnostic tool for prostatic tissue. A portable and compact multiphoton endoscope based on a 1-mm diameter, 8-cm length GRIN lens system probe was used. Fresh ex vivo samples were obtained from 14 radical prostatectomy patients and benign and malignant areas were imaged and correlated with subsequent H&E sections. Multiphoton GRIN endoscopy images of unfixed and unprocessed prostate tissue at a subcellular resolution are presented. We note several differences and identifying features of benign versus low-grade versus high-grade tumors and are able to identify periprostatic tissues such as adipocytes, periprostatic nerves, and blood vessels. Multiphoton GRIN endoscopy can be used to identify both benign and malignant lesions in ex vivo human prostate tissue and may be a valuable diagnostic tool for real-time visualization of suspicious areas of the prostate.

Linear and Nonlinear Viscoelastic Modeling of Aorta and Carotid Pressure-Area Dynamics under in vivo and ex vivo Conditions

PubMed Central

Valdez-Jasso, Daniela; Bia, Daniel; Zócalo, Yanina; Armentano, Ricardo L.; Haider, Mansoor A.; Olufsen, Mette S.

2013-01-01

A better understanding of the biomechanical properties of the arterial wall provides important insight into arterial vascular biology under normal (healthy) and pathological conditions. This insight has potential to improve tracking of disease progression and to aid in vascular graft design and implementation. In this study, we use linear and nonlinear viscoelastic models to predict biomechanical properties of the thoracic descending aorta and the carotid artery under ex vivo and in vivo conditions in ovine and human arteries. Models analyzed include a four-parameter (linear) Kelvin viscoelastic model and two five-parameter nonlinear viscoelastic models (an arctangent and a sigmoid model) that relate changes in arterial blood pressure to the vessel cross-sectional area (via estimation of vessel strain). These models were developed using the framework of Quasilinear Viscoelasticity (QLV) theory and were validated using measurements from the thoracic descending aorta and the carotid artery obtained from human and ovine arteries. In vivo measurements were obtained from ten ovine aortas and ten human carotid arteries. Ex vivo measurements (from both locations) were made in eleven male Merino sheep. Biomechanical properties were obtained through constrained estimation of model parameters. To further investigate the parameter estimates we computed standard errors and confidence intervals and we used analysis of variance to compare results within and between groups. Overall, our results indicate that optimal model selection depends on the arterial type. Results showed that for the thoracic descending aorta (under both experimental conditions) the best predictions were obtained with the nonlinear sigmoid model, while under healthy physiological pressure loading the carotid arteries nonlinear stiffening with increasing pressure is negligible, and consequently, the linear (Kelvin) viscoelastic model better describes the pressure-area dynamics in this vessel. Results

Liver volume measurement: reason of the difference between in vivo CT-volumetry and intraoperative ex vivo determination and how to cope it.

PubMed

Niehues, Stefan M; Unger, J K; Malinowski, M; Neymeyer, J; Hamm, B; Stockmann, M

2010-08-20

Volumetric assessment of the liver regularly yields discrepant results between pre- and intraoperatively determined volumes. Nevertheless, the main factor responsible for this discrepancy remains still unclear. The aim of this study was to systematically determine the difference between in vivo CT-volumetry and ex vivo volumetry in a pig animal model. Eleven pigs were studied. Liver density assessment, CT-volumetry and water displacement volumetry was performed after surgical removal of the complete liver. Known possible errors of volume determination like resection or segmentation borders were eliminated in this model. Regression analysis was performed and differences between CT-volumetry and water displacement determined. Median liver density was 1.07g/ml. Regression analysis showed a high correlation of r(2) = 0.985 between CT-volumetry and water displacement. CT-volumetry was found to be 13% higher than water displacement volumetry (p<0.0001). In this study the only relevant factor leading to the difference between in vivo CT-volumetry and ex vivo water displacement volumetry seems to be blood perfusion of the liver. The systematic difference of 13 percent has to be taken in account when dealing with those measures.

Ex Vivo Machine Perfusion in VCA with a Novel Oxygen Carrier System to Enhance Graft Preservation and Immunologic Outcomes

DTIC Science & Technology

2016-12-01

studies 2. Ex Vivo Study Group (n=4) Machine Perfusion 4 VRAM grafts preserved for 14 hours with MP at 21°C Study group – developments for machine...period. The VRAM grafts were inspected daily and punch biopsies were performed on days 2, 4 and 7. The end- study necropsy was conducted on post ...TNF- α in the MP group, showing the beneficial impact of effective ex -vivo oxygenation in this group (Figure 6). Previous clinical studies in

Proliferation of CD4CD25Foxp3 regulatory T lymphocytes in ex vivo expanded ascitic fluid from primary and recurrent ovarian carcinoma.

PubMed

Lee, Shin Wha; Kim, Yong-Man; Lee, Ha-Young; Kim, Dae-Yeon; Kim, Jong-Hyeok; Nam, Joo-Hyun; Kim, Young-Tak

2010-03-01

Regulatory T lymphocytes evoke the immune tolerance by suppressing and inactivating cytotoxic T lymphocytes. The objective of this study was to compare the proportion of regulatory T lymphocytes, precisely defined as CD4(+)CD25(high+)Foxp3(+) T lymphocytes, in primary and recurrent ovarian carcinoma before and after ex vivo expansion of ascites with interleukin-2 (IL-2). Ascitic fluid samples were obtained from 26 patients with ovarian carcinoma. Lymphocytes were isolated from ascites and cell markers were analyzed by flow cytometry using anti-CD3/CD4/CD8/CD16/CD56/CD25 and anti-Foxp3 antibodies. Lymphocytes were incubated for 2 to 3 weeks and expanded ex vivo by IL-2 stimulation and their phenotypes were analyzed by flow cytometry. Following ex vivo expansion, ascitic fluid lymphocytes increased by a greater extent in the recurrent group than in the primary group. The proportion of ex vivo-expanded lymphocytes changed as follows; CD4(+) T lymphocytes increased, CD8(+) T lymphocytes decreased, and the proportion of CD3(-)CD16(+)56(+) NK cells was unchanged. The proportion of CD4(+)CD25(high+)Foxp3(+) regulatory T lymphocytes in CD4(+) T lymphocytes increased after ex vivo expansion in both groups, but to a greater degree in the recurrent group. This study showed that regulatory T lymphocytes, neither cytotoxic T lymphocytes nor NK cells, were extensively increased after ex vivo expansion, especially in recurrent ovarian carcinoma. These results may provide information that helps to guide the future development of adoptive immunotherapy against ovarian carcinoma.

Investigation of allergenicity of some cosmetic mixtures by using ex vivo local lymph node assay-BrdU endpoints.

PubMed

Ulker, Ozge Cemiloglu; Kaymak, Yesim; Karakaya, Asuman

2014-01-01

Balsam of Peru and fragrance mix are commonly used in cosmetic products. Allergy to fragrance is the most common cause of cosmetic contact dermatitis. In the present study, ex vivo local lymph node assay-5-bromo-2'-deoxyuridine (LLNA-BrdU) was used to evaluate the dermal sensitization potential of these cosmetic mixtures. The stimulation index values and estimated concentration (EC3) values were calculated and the potency classification was found for each mixture. At the same time, in order to measure the irritant effect without having to use additional animals, a combination of ex vivo LLNA-BrdU and the irritancy assay was conducted. Th1 [interleukin (IL)-2, interferon-γ] and Th2 cytokine (IL-4, IL-5) releases from lymph node cell culture were investigated as non-radioactive endpoints. According to the results of ex vivo LLNA-BrdU assays, EC3 values were found to be 3.09% (moderate) for balsam of Peru and 4.44% (moderate) for fragrance mix. Cytokine analysis results indicate that both Th1 and Th2 cytokines are involved in the regulation of murine contact allergy and can be considered as useful endpoints. In conclusion, according to our results, fragrance mix and balsam of Peru can be considered as moderate sensitizers; however, in high concentrations, both of them have irritation properties. The cytokines investigated can be considered as the endpoints of the ex vivo LLNA-BrdU assay. © 2014 S. Karger AG, Basel.

Synthesis of conjugated chitosan and its effect on drug permeation from transdermal patches.

PubMed

Satheeshababu, B K; Shivakumar, K L

2013-03-01

The aim of this study was to synthesis the conjugated chitosan by covalent attachment of thiol moieties to the cationic polymer, mediated by a carbodiimide to improve permeation properties of chitosan. Thioglycolic acid was covalently attached to chitosan by the formation of amide bonds between the primary amino groups of the polymer and the carboxylic acid groups of thioglycolic acid. Hence, these polymers are called as thiomers or thiolated polymers. Conjugation of chitosan was confirmed by Fourier transform-infrared and differential scanning calorimetric analysis. Matrix type transdermal patches of carvedilol were prepared using the different proportions of chitosan and chitosan-thioglycolic acid conjugates (2:0, 1.7:0.3, 1.4:0.6, 1:1, 0.6:1.4 and 0.3:1.7) by solvent casting technique. Prepared matrix type patches were evaluated for their physicochemical characterization followed by in vitro evaluation. Selected formulations were subjected for their ex vivo studies on Wistar albino rat skin and human cadaver skin using the modified Franz diffusion cell. As the proportion of conjugated chitosan increased, the transdermal patches showed increased drug permeation. The mechanism of drug release was found to be nonFickian profiles. The present study concludes that the transdermal patches of carvedilol using conjugated chitosan with different proportions of chitosan were successfully developed to provide improved drug permeation. The transdermal patches can be a good approach to improve drug bioavailability by bypassing the extensive hepatic first-pass metabolism of the drug.

Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo

NASA Technical Reports Server (NTRS)

Fitzgerald, Wendy; Sylwester, Andrew W.; Grivel, Jean-Charles; Lifson, Jeffrey D.; Margolis, Leonid B.

2004-01-01

Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.

Evaluation of hybrid algorithm for analysis of scattered light using ex vivo nuclear morphology measurements of cervical epithelium

PubMed Central

Ho, Derek; Drake, Tyler K.; Bentley, Rex C.; Valea, Fidel A.; Wax, Adam

2015-01-01

We evaluate a new hybrid algorithm for determining nuclear morphology using angle-resolved low coherence interferometry (a/LCI) measurements in ex vivo cervical tissue. The algorithm combines Mie theory based and continuous wavelet transform inverse light scattering analysis. The hybrid algorithm was validated and compared to traditional Mie theory based analysis using an ex vivo tissue data set. The hybrid algorithm achieved 100% agreement with pathology in distinguishing dysplastic and non-dysplastic biopsy sites in the pilot study. Significantly, the new algorithm performed over four times faster than traditional Mie theory based analysis. PMID:26309741

Measurement of drug and macromolecule diffusion across atherosclerotic rabbit aorta ex vivo by attenuated total reflection-Fourier transform infrared imaging

NASA Astrophysics Data System (ADS)

Palombo, Francesca; Danoux, Charlène B.; Weinberg, Peter D.; Kazarian, Sergei G.

2009-07-01

Diffusion of two model drugs-benzyl nicotinate and ibuprofen-and the plasma macromolecule albumin across atherosclerotic rabbit aorta was studied ex vivo by attenuated total reflection-Fourier transform infrared (ATR-FTIR) imaging. Solutions of these molecules were applied to the endothelial surface of histological sections of the aortic wall that were sandwiched between two impermeable surfaces. An array of spectra, each corresponding to a specific location in the section, was obtained at various times during solute diffusion into the wall and revealed the distribution of the solutes within the tissue. Benzyl nicotinate in Ringer's solution showed higher affinity for atherosclerotic plaque than for apparently healthy tissue. Transmural concentration profiles for albumin demonstrated its permeation across the section and were consistent with a relatively low distribution volume for the macromolecule in the middle of the wall. The ability of albumin to act as a drug carrier for ibuprofen, otherwise undetected within the tissue, was demonstrated by multivariate subtraction image analysis. In conclusion, ATR-FTIR imaging can be used to study transport processes in tissue samples with high spatial and temporal resolution and without the need to label the solutes under study.

Porcine pulmonary auto-transplantation for ex vivo therapy as a model for new treatment strategies.

PubMed

Krüger, Marcus; Zinne, Norman; Biancosino, Christian; Höffler, Klaus; Rajab, Taufiek K; Waldmann, Karl-Heinz; Jonigk, Danny; Avsar, Murat; Haverich, Axel; Hoeltig, Doris

2016-09-01

Lung auto-transplantation is the surgical key step in experiments involving ex vivo therapy of severe or end-stage lung diseases. Ex vivo therapy has become a clinical reality because of systems such as the Organ Care System (OCS) Lung, which is the only commercially available portable lung perfusion system. However, survival experiments involving porcine lung auto-transplantation pose special surgical and anaesthesiological challenges. This current study was designed to describe the development of surgical techniques and aneasthesiological management strategies that facilitate lung auto-transplantation survival surgery including a follow-up period of 4 days. Left pneumonectomy was performed in 12 Mini-Lewe miniature pigs. After ex vivo treatment of the harvested lungs within the OCS Lung for 2 h, the lungs were retransplanted into the same animal (auto-transplantation). Four animals were used to develop the optimal techniques and establish an experimental protocol. According to the final protocol, eight additional animals were operated. The follow-up period was 4 days. There were four severe intraoperative surgical complications [anatomical variant of the superior vena cava (two times), a complication related to the bronchial anastomosis and a complication related to the pulmonary arterial anastomosis]. The major postoperative problems were hyperkalaemia, prolonged recovery from anaesthesia and pulmonary oedema after reperfusion. Establishment of the surgical technique showed that using a pericardial tube to facilitate the anastomosis of the thin left superior pulmonary vein should be considered to prevent thrombosis. However, routine use of the patch technique to construct venous and arterial anastomoses is not necessary. Furthermore, traction on the venous anastomoses can be avoided by performing the bronchial anastomosis first. Lung auto-transplantation is a feasible experimental model for ex vivo therapy of lung diseases and is applicable for experimental

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

PubMed Central

Zhang, Jun; Preda, Dorin V.; Vasquez, Kristine O.; Morin, Jeff; Delaney, Jeannine; Bao, Bagna; Percival, M. David; Xu, Daigen; McKay, Dan; Klimas, Michael; Bednar, Bohumil; Sur, Cyrille; Gao, David Z.; Madden, Karen; Yared, Wael; Rajopadhye, Milind

2012-01-01

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments. PMID:22674025

Development of a rate model to investigate contributions of anatomic and physiologic determinants of in vivo skin permeation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fleischer, N.M.

The skin is a heterogeneous, bi-directional impediment to chemical flux, in which the stratum corneum is a major, though not the sole, rate-limiting barrier layer to permeation. Systemic toxicity following dermal exposure to environmental chemicals and use of skin as a portal for systemic administration of drugs have led to extensive investigations of the inward flux of xenobiotics applied to the outer surface of skin. Those investigations mainly utilized in vitro experimental systems that were limited by the absence of normal physiologic functions. The objective of the present research was to investigate an in vivo skin permeation model system thatmore » was sensitive to perturbations of skin capillary physiology and stratum corneum. A [open quotes]fuzzy[close quotes] rat model system was devised that employed outward cutaneous migration of a systemically administered permeation probe, isoflurane. Specially devised, transdermal vapor collection devices were used to capture the outward flux of isoflurane through the skin. Isoflurane flux measurements, coupled with blood isoflurane concentrations, were used to calculate cutaneous permeability coefficients (K[sub p]) of isolflurane, as an index of permeation, under various conditions of normal or perturbed cutaneous physiologic states. Physiologic perturbations were performed to test the sensitivity of the model system to detect effects of minoxidil-mediated vasodilation, phenylephrine-mediated vasoconstriction, and leukotriene D[sub 4]-mediated increased capillary permeability on the outward flux of isoflurane. Tape stripping and topical ether-ethanol application produced either physical removal or chemical disruption of the stratum corneum, respectively. Minoxidil, leukotriene D[sub 4], tape stripping of stratum corneum, and topical ether-ethanol experiments produced statistically significant increases (52 to 193%) in the K[sub p's], while phenylephrine had no significant effect on isoflurane permeation.« less

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo.

PubMed

Altenburg, Arwen F; van de Sandt, Carolien E; Li, Bobby W S; MacLoughlin, Ronan J; Fouchier, Ron A M; van Amerongen, Geert; Volz, Asisa; Hendriks, Rudi W; de Swart, Rik L; Sutter, Gerd; Rimmelzwaan, Guus F; de Vries, Rory D

2017-08-17

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.

Ex vivo imaging and quantification of liver fibrosis using second-harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Sun, Tzu-Lin; Liu, Yuan; Sung, Ming-Chin; Chen, Hsiao-Ching; Yang, Chun-Hui; Hovhannisyan, Vladimir; Lin, Wei-Chou; Jeng, Yung-Ming; Chen, Wei-Liang; Chiou, Ling-Ling; Huang, Guan-Tarn; Kim, Ki-Hean; So, Peter T. C.; Chen, Yang-Fang; Lee, Hsuan-Shu; Dong, Chen-Yuan

2010-05-01

Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.

Ex vivo imaging and quantification of liver fibrosis using second-harmonic generation microscopy.

PubMed

Sun, Tzu-Lin; Liu, Yuan; Sung, Ming-Chin; Chen, Hsiao-Ching; Yang, Chun-Hui; Hovhannisyan, Vladimir; Lin, Wei-Chou; Jeng, Yung-Ming; Chen, Wei-Liang; Chiou, Ling-Ling; Huang, Guan-Tarn; Kim, Ki-Hean; So, Peter T C; Chen, Yang-Fang; Lee, Hsuan-Shu; Dong, Chen-Yuan

2010-01-01

Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.

Gravitational physiology of human immune cells: a review of in vivo, ex vivo and in vitro studies

NASA Technical Reports Server (NTRS)

Cogoli, A.

1996-01-01

The study of the function of immune cells in microgravity has been studied for more than 20 years in several laboratories. It is clear today that the immune system is depressed in more than 50% of the astronauts during and after space flight and that the activation of T lymphocytes by mitogens in vitro changes dramatically. This article gives an overview of the gravitational studies conducted by our laboratory in Spacelab, in MIR station, in sounding rockets and on the ground in the clinostat and the centrifuge. Three experimental approaches are followed in our work: (i) Ex vivo studies are performed with blood samples drawn from astronauts; (ii) in vivo studies are based on the application of seven antigens to the skin of the astronauts; (iii) in vitro studies are carried out with immune cells purified from the blood of healthy donors (not astronauts). The data from our in vivo and ex vivo studies are in agreement with those of other laboratories and show that the immunological function is depressed in the majority of astronauts as a consequence of the stress of space flight rather than by a direct influence of gravity on the cell. Immune depression may become a critical hazard on long duration flights on space stations or to other planets. In vitro experiments show that cultures of free-floating lymphocytes and monocytes undergo a dramatic depression of activation by the mitogen concanavalin A, while activation is more than doubled when the cells are attached to microcarrier beads. Such effects may be attributed to both direct and indirect effects of gravitational unloading on basic biological mechanisms of the cell. While the in vitro data are very important to clarify certain aspects of the biological mechanism of T cells activation, they are not descriptive of the changes of the immunological function of the astronauts.

Gravitational physiology of human immune cells: a review of in vivo, ex vivo and in vitro studies.

PubMed

Cogoli, A

1996-04-01

The study of the function of immune cells in microgravity has been studied for more than 20 years in several laboratories. It is clear today that the immune system is depressed in more than 50% of the astronauts during and after space flight and that the activation of T lymphocytes by mitogens in vitro changes dramatically. This article gives an overview of the gravitational studies conducted by our laboratory in Spacelab, in MIR station, in sounding rockets and on the ground in the clinostat and the centrifuge. Three experimental approaches are followed in our work: (i) Ex vivo studies are performed with blood samples drawn from astronauts; (ii) in vivo studies are based on the application of seven antigens to the skin of the astronauts; (iii) in vitro studies are carried out with immune cells purified from the blood of healthy donors (not astronauts). The data from our in vivo and ex vivo studies are in agreement with those of other laboratories and show that the immunological function is depressed in the majority of astronauts as a consequence of the stress of space flight rather than by a direct influence of gravity on the cell. Immune depression may become a critical hazard on long duration flights on space stations or to other planets. In vitro experiments show that cultures of free-floating lymphocytes and monocytes undergo a dramatic depression of activation by the mitogen concanavalin A, while activation is more than doubled when the cells are attached to microcarrier beads. Such effects may be attributed to both direct and indirect effects of gravitational unloading on basic biological mechanisms of the cell. While the in vitro data are very important to clarify certain aspects of the biological mechanism of T cells activation, they are not descriptive of the changes of the immunological function of the astronauts.

ACE2-Independent Action Of Presumed ACE2 Activators: Studies In Vivo, Ex Vivo and In Vitro

PubMed Central

Haber, Philipp K.; Ye, Minghao; Wysocki, Jan; Maier, Christoph; Haque, Syed K.; Batlle, Daniel

2014-01-01

Angiotensin converting enzyme 2, (ACE2), is a key enzyme in the metabolism of angiotensin II. 1-[[2-(dimetilamino)ethyl]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT)and Diminazene (DIZE)have been reported to exert various organ-protective effects that have been attributed to activation of ACE2. To test the effect of these compounds we studied Ang II degradation in vivo and in vitro as well as their effect on ACE2 activity in vivo and in vitro. In a model of Ang II induced acute hypertension, blood pressure recovery was markedly enhanced by XNT (slope with XNT -3.26±0.2 vs.-1.6±0.2 mmHg/min without XNT, p<0.01). After Ang II infusion, neither plasma nor kidney ACE2 activity was affected by XNT. Plasma Ang II and Ang (1-7) levels also were not significantly affected by XNT. The blood pressure lowering effect of XNT seen in WT animals was also observed in ACE2 KO mice (slope with XNT -3.09±0.30 mmHg/min vs. -1.28±0.22 mmHg/min without XNT, p<0.001). These findings show that the blood pressure lowering effect of XNT in Ang II induced hypertension cannot be due to activation of ACE2. In vitro and ex vivo experiments in both mice and rat kidney confirmed a lack of enhancement of ACE2 enzymatic activity by XNT and DIZE. Moreover, Ang II degradation in vitro and ex vivo was unaffected by XNT and DIZE. We conclude that the biologic effects of these compounds are ACE2 independent and should not be attributed to activation of this enzyme. PMID:24446061

Imaging in vivo secondary caries and ex vivo dental biofilms using cross-polarization optical coherence tomography

PubMed Central

Lenton, Pat; Rudney, Joel; Chen, Ruoqiong; Fok, Alex; Aparicio, Conrado; Jones, Robert S.

2012-01-01

Objectives Conventional diagnostic methods frequently detect only late stage enamel demineralization under composite resin restorations. The objective of this study is to examine the subsurface tooth-composite interface and to assess for the presence of secondary caries in pediatric patients using a novel Optical Coherence Tomography System with an intraoral probe. Methods A newly designed intraoral cross polarization swept source optical coherence tomography (CP-OCT) imaging system was used to examine the integrity of the enamel-composite interfaces in vivo. Twenty two pediatric subjects were recruited with either recently placed or long standing composite restorations in their primary teeth. To better understand how bacterial biofilms cause demineralization at the interface, we also used the intraoral CP-OCT system to assess ex vivo bacterial biofilm growth on dental composites. Results As a positive control, cavitated secondary carious interfaces showed a 18.2 dB increase (p<0.001), or over 1-2 orders of magnitude higher, scattering than interfaces associated with recently placed composite restorations. Several long standing composite restorations, which appeared clinically sound, had a marked increase in scattering than recently placed restorations. This suggests the ability of CP-OCT to assess interfacial degradation such as early secondary caries prior to cavitation. CP-OCT was also able to image ex vivo biofilms on dental composites and assess their thickness. Significance This paper shows that CP-OCT imaging using a beam splitter based design can examine the subsurface interface of dental composites in human subjects. Furthermore, the probe dimensions and acquisition speed of the CP-OCT system allowed for analysis of caries development in children. PMID:22578989

Evaluation of ex-vivo 9.4T MRI in post-surgical specimens from temporal lobe epilepsy patients.

PubMed

Kwan, Benjamin Y M; Salehi, Fateme; Kope, Ryan; Lee, Donald H; Sharma, Manas; Hammond, Robert; Burneo, Jorge G; Steven, David; Peters, Terry; Khan, Ali R

2017-10-01

This study evaluates hippocampal pathology through usage of ultra-high field 9.4T ex-vivo imaging of resected surgical specimens in patients who have undergone temporal lobe epilepsy surgery. This is a retrospective interpretation of prospectively acquired data. MRI scanning of resected surgical specimens from patients who have undergone temporal lobe epilepsy surgery was performed on a 9.4T small bore Varian MR magnet. Structural images employed a balanced steady-state free precession sequence (TrueFISP). Six patients (3 females; 3 males) were included in this study with an average age at surgery of 40.7 years (range 20Y_"60) (one was used as a control reference). Two neuroradiologists qualitatively reviewed the ex-vivo MRIs of resected specimens while blinded to the histopathology reports for the ability to identify abnormal features in hippocampal subfield structures. The hippocampal subfields were reliably identified on the 9.4T ex-vivo scans in the hippocampal head region and hippocampal body region by both neuroradiologists in all 6 patients. There was high concordance to pathology for abnormalities detected in the CA1, CA2, CA3 and CA4 subfields. Detection of abnormalities in the dentate gyrus was also high with detection in 4 of 5 cases. The Cohen's kappa between the two neuroradiologists was calculated at 0.734 SE=0.102. Ex-vivo 9.4T specimen imaging can detect abnormalities in CA1, CA2, CA3, CA4 and DG in both the hippocampal head and body. There was good concordance between qualitative findings and histopathological abnormalities for CA1, CA2, CA3, CA4 and DG. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis.

PubMed

Pinnock, Abigail; Shivshetty, Nagaveni; Roy, Sanhita; Rimmer, Stephen; Douglas, Ian; MacNeil, Sheila; Garg, Prashant

2017-02-01

In the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models. Excised rabbit and human corneoscleral rims maintained in organ culture were infected using 10 8 cells of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans or Fusarium solani. The infection was introduced by wounding with a scalpel and exposing corneas to the microbial suspension or by intrastromal injection. Post-inoculation, corneas were maintained for 24 and 48 h at 37 °C. After incubation, corneas were either homogenised to determine colony-forming units (CFU)/cornea or processed for histological examination using routine staining methods. Single- and mixed-species infections were compared. We observed a significant increase in CFU after 48 h compared to 24 h with S. aureus and P. aeruginosa. However, no such increase was observed in corneas infected with C. albicans or F. solani. The injection method yielded an approximately two- to 100-fold increase (p < 0.05) in the majority of organisms from infected corneas. Histology of the scalpel-wounded and injection models indicated extensive infiltration of P. aeruginosa throughout the entire cornea, with less infiltration observed for S. aureus, C. albicans and F. solani. The models also supported dual infections. Both scalpel wounding and injection methods are suitable for inducing infection of ex vivo rabbit and human cornea models. These simple and reproducible models will be useful as an alternative to in vitro and in vivo models for investigating the detection and treatment of microbial keratitis, particularly when this might be due to two infective organisms.

A non-covalent peptide-based strategy for ex vivo and in vivo oligonucleotide delivery.

PubMed

Crombez, Laurence; Morris, May C; Heitz, Frederic; Divita, Gilles

2011-01-01

The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

Novel ex vivo culture method for human monocytes uses shear flow to prevent total loss of transendothelial diapedesis function.

PubMed

Tsubota, Yoshiaki; Frey, Jeremy M; Raines, Elaine W

2014-01-01

Monocyte recruitment to inflammatory sites and their transendothelial migration into tissues are critical to homeostasis and pathogenesis of chronic inflammatory diseases. However, even short-term suspension culture of primary human monocytes leads to phenotypic changes. In this study, we characterize the functional effects of ex vivo monocyte culture on the steps involved in monocyte transendothelial migration. Our data demonstrate that monocyte diapedesis is impaired by as little as 4 h culture, and the locomotion step is subsequently compromised. After 16 h in culture, monocyte diapedesis is irreversibly reduced by ∼90%. However, maintenance of monocytes under conditions mimicking physiological flow (5-7.5 dyn/cm²) is sufficient to reduce diapedesis impairment significantly. Thus, through the application of shear during ex vivo culture of monocytes, our study establishes a novel protocol, allowing functional analyses of monocytes not currently possible under static culture conditions. These data further suggest that monocyte-based therapeutic applications may be measurably improved by alteration of ex vivo conditions before their use in patients.

Fast isolation and ex vivo culture of circulating tumor cells from the peripheral blood of lung cancer patients.

PubMed

Wu, Wen-jun; Wang, Zhi-hua; Wang, Zhuo; Deng, Yu-liang; Shi, Qi-hui

2017-01-20

Circulating tumor cells (CTCs) are free tumor cells shed from tumor site and enter into blood circulation. CTCs represent a reliable source of tumor cells for the molecular characteristics of the original tumor. However, the extraordinary rarity of CTCs makes the subsequent molecular and functional analysis technically challenging. Here, we describe a one-step microfludics-based immunomagnetic isolation method to isolate CTCs directly from the whole blood of lung adenocarcinoma patients. This method avoids harsh sample preparation and enrichment steps, and therefore preserves the viability (>90%) of CTCs during the in vitro isolation. The isolated CTCs are enriched in small volume (80 μL) and cultured ex vivo that leads to successful ex vivo expansion. The expanded CTCs can be frozen and thawed, which shows cell line property. Genetic sequencing on EGFR、KRAS、PIK3CA、TP53 and BRAF and metabolic assay (2-NBDG) are utilized to characterize the expanded CTCs. Our results demostrated that this method is suitable for ex vivo expansion of CTCs facilitates. The genomic, proteomic and metabolic analyses of CTCs have guiding significance in tumor precise treatment.

Fluorescence Confocal Microscopy for Ex Vivo Diagnosis of Conjunctival Tumors: A Pilot Study.

PubMed

Iovieno, Alfonso; Longo, Caterina; De Luca, Mariacarla; Piana, Simonetta; Fontana, Luigi; Ragazzi, Moira

2016-08-01

To evaluate the potential use of fluorescence confocal microscopy (FCM) for ex vivo diagnosis and excision margin assessment of conjunctival neoplasms. Validity study. setting: Single institution. Consecutive patients with clinically suspicious conjunctival lesions. Conjunctival lesions were excised in toto using a standard "no-touch technique" by a single surgeon (A.I.). Collected specimens were examined with a commercially available laser scanning fluorescence confocal microscope after immersion in a 0.6 mM solution of acridine orange dye for 10-20 seconds. Specimens were subsequently processed with standard histologic analysis. FCM diagnosis of the nature and extension of conjunctival lesions. Sixteen consecutive patients were included in the study (11 male, 5 female; mean age 58.1 ± 26.1 years, range 10-90 years). The median time needed to process and analyze a sample with FCM was 15 minutes. Eleven of 16 lesions were identified by FCM as squamous (2 benign papillomas, 2 grade 2 conjunctival intraepithelial neoplasias, 7 in situ squamous carcinomas) and 5 as nonsquamous (1 pingueculum, 1 dermolipoma, 2 melanocytic nevi, 1 melanoma). In all cases FCM was able to detect horizontal and vertical extension of the lesion. All FCM findings were confirmed by corresponding subsequent histologic examination. FCM provides a fast ex vivo preliminary diagnosis of suspicious conjunctival lesions with good histologic details and margin assessment, and may represent a novel tool for intraoperative and postsurgical management of conjunctival tumors. This is the first study to investigate ex vivo FCM application in ophthalmology. Copyright © 2016 Elsevier Inc. All rights reserved.

Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

PubMed

Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

2014-02-01

Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P < 0·001). The results demonstrate the feasibility of confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

Intra-patient variability of thromboelastographic parameters following in vivo and ex vivo administration of recombinant activated factor VII in haemophilia patients. A multi-centre, randomised trial.

PubMed

Kenet, G; Stenmo, C B; Blemings, A; Wegert, W; Goudemand, J; Krause, M; Schramm, W; Kirchmaier, C; Martinowitz, U

2010-02-01

Thromboelastography methods have been used to predict or monitor treatment of haemophilia patients with recombinant activated factor VII (rFVIIa). However, neither of the two thromboelastographic methods (ROTEM and TEG) has as yet been validated. This multi-centre, randomised trial compared both methods in terms of intra- and inter- patient variability following in vivo and ex vivo rFVIIa administration to haemophilia A and B patients with and without inhibitors. Patients ((3)16 years old) received the same intravenous rFVIIa dose (45, 90 or 180 microg/kg) twice, 1-12 weeks apart. Blood samples were collected pre-dose and 15, 60, 120 and 240 minutes post-dose for ROTEM and TEG analysis. Pre-dose samples were also spiked ex vivo with rFVIIa (0.6, 1.2 or 2.4 microg/ml), to correspond to the three in vivo doses. Twenty-six haemophilia A and four haemophilia B patients were enrolled. A significant treatment effect was observed with in vivo rFVIIa (p<0.05) with more pronounced effects in inhibitor (n=14) versus non-inhibitor (n=16) patients. There was a strong positive correlation between ROTEM and TEG parameters. Intra- and inter-patient variation was large for all thromboelastography parameters at all time points and rFVIIa doses. Intra-patient variation was generally lower for non-inhibitor than inhibitor patients, and lower following ex vivo spiking versus in vivo rFVIIa administration. In conclusion, there was a clear effect of rFVIIa on all thromboelastography parameters, but the large intra- and inter-patient variability following in vivo rFVIIa administration renders the use of our method unsuitable for dose-response prediction for haemophilia patients in the clinical setting.

Helicobacter pylori-induced gastric pathology: insights from in vivo and ex vivo models.

PubMed

Burkitt, Michael D; Duckworth, Carrie A; Williams, Jonathan M; Pritchard, D Mark

2017-02-01

Gastric colonization with Helicobacter pylori induces diverse human pathological conditions, including superficial gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma and its precursors. The treatment of these conditions often relies on the eradication of H. pylori, an intervention that is increasingly difficult to achieve and that does not prevent disease progression in some contexts. There is, therefore, a pressing need to develop new experimental models of H. pylori-associated gastric pathology to support novel drug development in this field. Here, we review the current status of in vivo and ex vivo models of gastric H. pylori colonization, and of Helicobacter-induced gastric pathology, focusing on models of gastric pathology induced by H. pylori, Helicobacter felis and Helicobacter suis in rodents and large animals. We also discuss the more recent development of gastric organoid cultures from murine and human gastric tissue, as well as from human pluripotent stem cells, and the outcomes of H. pylori infection in these systems. © 2017. Published by The Company of Biologists Ltd.

A Novel Ex Vivo Model to Investigate the Underlying Mechanisms in Alzheimer’s Disease

PubMed Central

Brai, Emanuele; Stuart, Skye; Badin, Antoine-Scott; Greenfield, Susan A.

2017-01-01

Currently there is no widely accepted animal model reproducing the full pathological profile of Alzheimer’s disease (AD), since the basic mechanisms of neurodegeneration are still poorly understood. We have proposed that the interaction between the α7 nicotinic acetylcholine receptor (α7-nAChR) and a recently discovered toxic peptide, cleaved from the acetylcholinesterase (AChE) C-terminus, could account for the aberrant processes occurring in AD. In this article we describe a new application on ex vivo model procedure, which combines the advantages of both in vivo and in vitro preparations, to study the effects of the AChE-derived peptide on the rat basal forebrain (BF). Western blot analysis showed that the levels of α7-nAChR, p-Tau and Aβ are differentially expressed upon the AChE-peptide administration, in a selective site-dependent manner. In conclusion, this methodology demonstrates the action of a novel peptide in triggering an AD-like phenotype and proposes a new ex vivo approach for manipulating and monitoring neurochemical processes contributing to neurodegeneration, in a time-dependent and site-specific manner. PMID:29033787

Ex vivo T2 relaxation: Associations with age-related neuropathology and cognition

PubMed Central

Dawe, Robert J.; Bennett, David A.; Schneider, Julie A.; Leurgans, Sue E.; Kotrotsou, Aikaterini; Boyle, Patricia A.; Arfanakis, Konstantinos

2014-01-01

The transverse relaxation time constant, T2, is sensitive to brain tissue’s free water content and the presence of paramagnetic materials such as iron. In this study, ex vivo MRI was employed to investigate alterations in T2 related to Alzheimer’s disease (AD) pathology and other types of neuropathology common in old age, as well as the relationship between T2 alterations and cognition. Cerebral hemispheres were obtained from 371 deceased older adults. Using fast spin-echo imaging with multiple echo times, T2 maps were produced and warped to a study-specific template. Hemispheres underwent neuropathologic examination for identification of AD pathology and other common age-related neuropathologies. Voxelwise linear regression was carried out to detect regions of pathology-related T2 alterations and, in separate analyses, regions in which T2 alterations were linked to antemortem cognitive performance. AD pathology was associated with T2 prolongation in white matter of all lobes and T2 shortening in the basal ganglia and insula. Gross infarcts were associated with T2 prolongation in white matter of all lobes, and in the thalamus and basal ganglia. Hippocampal sclerosis was associated with T2 prolongation in the hippocampus and white matter of the temporal lobe. After controlling for neuropathology, T2 prolongation in the frontal lobe white matter was associated with lower performance in the episodic, semantic, and working memory domains. In addition, voxelwise analysis of in vivo and ex vivo T2 values indicated a positive relationship between the two, though further investigation is necessary to accurately translate findings of the current study to the in vivo case. PMID:24582637

Temperature changes in dental implants following exposure to hot substances in an ex vivo model.

PubMed

Feuerstein, Osnat; Zeichner, Kobi; Imbari, Chen; Ormianer, Zeev; Samet, Nachum; Weiss, Ervin I

2008-06-01

The habitual consumption of extremely hot foods and beverages may affect implant treatment modality. Our objectives were to: (i) establish the maximum temperature produced intra-orally while consuming very hot substances and (ii) use these values in an ex vivo model to assess the temperature changes along the implant-bone interface. Temperatures were measured using thermocouples linked to a computer. The thermocouple electrodes were attached to the tooth-gum interface of the interproximal areas in 14 volunteers during consumption of extremely hot foods and beverages. The in vivo measured temperature values obtained were used in an ex vivo model of a bovine mandible block with an implant and with an assembled abutment. Temperatures were measured by thermocouple electrodes attached to five locations, three of them along the implant-bone interface. During consumption of a hot beverage, a maximum temperature of up to 76.3 degrees C was recorded, and a calculated extreme intra-oral temperature of 61.4 degrees C was established. The ex vivo model showed a high correlation between the temperature measured at the abutment and that measured at the abutment-implant interface and inside the implant, reaching maximum temperatures close to 60 degrees C. At the mid-implant-bone and apical implant-bone interfaces, the maximum temperatures measured were 43.3 and 42 degrees C, respectively. The maximum temperatures measured at the implant-bone interfaces reached the temperature threshold of transient changes in bone (42 degrees C). The results of this study support the notion that intra-oral temperatures, developed during the consumption of very hot substances, may be capable of damaging peri-implant tissues.

In vitro-ex vivo correlations between a cell-laden hydrogel and mucosal tissue for screening composite delivery systems

PubMed Central

Blakney, Anna K.; Little, Adam B.; Jiang, Yonghou; Woodrow, Kim A.

2017-01-01

Composite delivery systems where drugs are electrospun in different layers and vary the drug stacking-order are posited to affect bioavailability. We evaluated how the formulation characteristics of both burst- and sustained-release electrospun fibers containing three physicochemically diverse drugs: dapivirine (DPV), maraviroc (MVC) and tenofovir (TFV) affect in vitro and ex vivo release. We developed a poly(hydroxyethyl methacrylate) (pHEMA) hydrogel release platform for the rapid, inexpensive in vitro evaluation of burst- and sustained-release topical or dermal drug delivery systems with varying microarchitecture. We investigated properties of the hydrogel that could recapitulate ex vivo release into nonhuman primate vaginal tissue. Using a DMSO extraction protocol and HPLC analysis, we achieved >93% recovery from the hydrogels and >88% recovery from tissue explants for all three drugs. We found that DPV loading, but not stacking order (layers of fiber containing a single drug) or microarchitecture (layers with isolated drug compared to all drugs in the same layer) impacted the burst release in vitro and ex vivo. Our burst-release formulations showed a correlation for DPV accumulation between the hydrogel and tissue (R2=0.80), but the correlation was not significant for MVC or TFV. For the sustained release formulations, the PLGA/PCL content did not affect TFV release in vitro or ex vivo. Incorporation of cells into the hydrogel matrix improved the correlation between hydrogel and tissue explant release for TFV. We expect that this hydrogel tissue mimic maybe a promising preclinical model to evaluate topical or transdermal drug delivery systems with complex microarchitectures. PMID:28222612

In vitro-ex vivo correlations between a cell-laden hydrogel and mucosal tissue for screening composite delivery systems.

PubMed

Blakney, Anna K; Little, Adam B; Jiang, Yonghou; Woodrow, Kim A

2016-11-01

Composite delivery systems where drugs are electrospun in different layers and vary the drug stacking-order are posited to affect bioavailability. We evaluated how the formulation characteristics of both burst- and sustained-release electrospun fibers containing three physicochemically diverse drugs: dapivirine (DPV), maraviroc (MVC) and tenofovir (TFV) affect in vitro and ex vivo release. We developed a poly(hydroxyethyl methacrylate) (pHEMA) hydrogel release platform for the rapid, inexpensive in vitro evaluation of burst- and sustained-release topical or dermal drug delivery systems with varying microarchitecture. We investigated properties of the hydrogel that could recapitulate ex vivo release into nonhuman primate vaginal tissue. Using a dimethyl sulfoxide extraction protocol and high-performance liquid chromatography analysis, we achieved >93% recovery from the hydrogels and >88% recovery from tissue explants for all three drugs. We found that DPV loading, but not stacking order (layers of fiber containing a single drug) or microarchitecture (layers with isolated drug compared to all drugs in the same layer) impacted the burst release in vitro and ex vivo. Our burst-release formulations showed a correlation for DPV accumulation between the hydrogel and tissue (R 2 =   0.80), but the correlation was not significant for MVC or TFV. For the sustained-release formulations, the PLGA/PCL content did not affect TFV release in vitro or ex vivo. Incorporation of cells into the hydrogel matrix improved the correlation between hydrogel and tissue explant release for TFV. We expect that this hydrogel-tissue mimic may be a promising preclinical model to evaluate topical or transdermal drug delivery systems with complex microarchitectures.

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in CD34+CD133+CXCR4low fraction.

PubMed

Lapostolle, Véronique; Chevaleyre, Jean; Duchez, Pascale; Rodriguez, Laura; Vlaski-Lafarge, Marija; Sandvig, Ioanna; Brunet de la Grange, Philippe; Ivanovic, Zoran

2018-06-01

Feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, CD34, CD133, CD90, CD45RA, CD26 and CD9 expression was determined on sorted CD34+ cells according to CXCR4 (neg, low, bright) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained in only the CD133+CXCR4low cells. The failure of CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood ones, as well as to those issued from the bone marrow. This data represent the first phenotypic characterization of steady-state blood cells exhibiting short and long term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation. Copyright © 2018, Ferrata Storti Foundation.

PREPARATION, IN VITRO AND IN VIVO CHARACTERIZATION OF HYDROPHOBIC PATCHES OF A HIGHLY WATER SOLUBLE DRUG FOR PROLONGED PLASMA HALF LIFE: EFFECT OF PERMEATION ENHANCERS.

PubMed

Yaqoob, Ayesha; Ahmad, Mahmood; Mahmood, Asif; Sarfraz, Rai Muhammad

2016-11-01

Aim of present study was to develop metoprolol matrix patches using different enhancers. Combination of two hydrophobic polymers, ethyl cellulose and eudragit RL 100 (8 : 2) were used for preparation of unilaminated matrix patch. 10% w/w of isopropyl myristate (IPM), dimethyl sulfoxide (DMSO), span (20 (S20), Tween 20 (T20) and eucalyptus oil as enhancers and 40% of dibutyl phthalate as plasticizer were used. Prepared patches were evaluated for physical appearance, weight uniformity and thickness. FTIR studies were performed to assess compatibility among ingredients and developed formulation. Dissolution and permeation studies were performed to compare effects of enhancers. Surface morphology after release was examined by scanning electron microscopy. Selected formulation was subjected to in vivo studies by randomized crossover design in rabbits (n = 6) for pharmacokinetic comparison with oral solution administration. Physical evaluation revealed that translucent, flexible, non brittle patches of uniform weight and thickness were prepared. Release from patches followed Higuchi model. Mechanism of release was Fickian. Formulation containing IPM showed that release was by anomalous transport. Highest permeation flux was observed for formulation containing IPM with 2-fold enhancement in permeation. Permeation flux for patches was in order of formulation with no enhancer > IPM > T20 > S20 > DMSO = eucalyptus oil. Plasma concentration from in vivo studies exhibited sustained plasma levels of metoprolol after transdermal patch application in comparison to oral solution administration. Pharmacokinetic analysis of in vivo data elucidated that half life was increased 8 times when compared to oral administration, due to controlled release of drug for longer period of time. These findings suggested that hydrophobic transdermal patches of highly water soluble drug metoprolol were successfully prepared with 10% of IPM for sustained systemic delivery for prolonged half life.

Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers.

PubMed

Williams, W C; Copeland, C; Boykin, E; Quell, S J; Lehmann, D M

2015-01-01

The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [(3) H]methyl thymidine. A more recent non-isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non-sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU-enzyme-linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis-diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC2 values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Ex vivo MRI evaluation of prostate cancer: Localization and margin status prediction of prostate cancer in fresh radical prostatectomy specimens.

PubMed

Heidkamp, Jan; Hoogenboom, Martijn; Kovacs, Iringo E; Veltien, Andor; Maat, Arie; Sedelaar, J P Michiel; Hulsbergen-van de Kaa, Christina A; Fütterer, Jurgen J

2018-02-01

To investigate the ability of high field ex vivo magnetic resonance imaging (MRI) to localize prostate cancer (PCa) and to predict the margin status in fresh radical prostatectomy (RP) specimens using histology as the reference standard. This Institutional Review Board (IRB)-approved study had written informed consent. Patients with biopsy-proved PCa and a diagnostic multiparametric 3T MRI examination of the prostate prior to undergoing RP were prospectively included. A custom-made container provided reference between the 7T ex vivo MRI obtained from fresh RP specimens and histological slicing. On ex vivo MRI, PCa was localized and the presence of positive surgical margins was determined in a double-reading session. These findings were compared with histological findings obtained from completely cut, whole-mount embedded, prostate specimens. In 12 RP specimens, histopathology revealed 36 PCa lesions, of which 17 (47%) and 20 (56%) were correlated with the ex vivo MRI in the first and second reading session, respectively. Nine of 12 (75%) index lesions were localized in the first session, in the second 10 of 12 (83%). Seven and 8 lesions of 11 lesions with Gleason score >6 and >0.5 cc were localized in the first and second session, respectively. In the first session none of the four histologically positive surgical margins (sensitivity 0%) and 9 of 13 negative margins (specificity 69%) were detected. In second session the sensitivity and specificity were 25% and 88%, respectively. Ex vivo MRI enabled accurate localization of PCa in fresh RP specimens, and the technique provided information on the margin status with high specificity. 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:439-448. © 2017 International Society for Magnetic Resonance in Medicine.

Evaluation of non-radioactive endpoints of ex vivo local lymph node assay-BrdU to investigate select contact sensitizers.

PubMed

Ulker, Ozge Cemiloglu; Ates, Ilker; Atak, Aysegul; Karakaya, Asuman

2013-01-01

The present study sought to verify the utility of the non-radioactive endpoints LLNA BrdU (5-bromo-2'-deoxyuridine) ex vivo incorporation and cytokine release using auricular lymph node cells isolated from BALB/c mice topically treated with a strong (formaldehyde or p-phenylene-diamine [PPD]), moderate sensitizer (cinnamal), or weak sensitizer (eugenol). Stimulation index (SI) and EC₃ values were calculated for each agent. Based on the results of ex vivo LLNA-BrdU assays, EC₃ values were calculated to be 0.29, 0.09, 1.91, and 16.60% for formaldehyde, PPD, cinnamal, and eugenol, respectively. These results were in good agreement with data from previous standard radioactive LLNA. Cytokine analyses indicated T(H)1 and T(H)2 cytokine involvement in the regulation of murine contact allergy and these could be utilized as endpoints in assessments of contact allergy in mice. In conclusion, the current study provided evidence that the non-radioactive endpoint LLNA BrdU ex vivo incorporation could be of use as a viable alternative approach to assess the skin sensitization potential of test compound with respect to improving animal welfare. This is of particular importance in the case of any laboratory where it might be difficult to handle and/or readily employ radioisotopes. Further studies will be required to confirm--across test agents--the reproducibility as well as the limits of utility of this new ex vivo BrdU method.

Monitoring Dopamine ex Vivo during Electrical Stimulation Using Liquid-Microjunction Surface Sampling.

PubMed

Gill, Emily L; Marks, Megan; Yost, Richard A; Vedam-Mai, Vinata; Garrett, Timothy J

2017-12-19

Liquid-microjunction surface sampling (LMJ-SS) is an ambient ionization technique based on the continuous flow of solvent using an in situ microextraction device in which solvent moves through the probe, drawing in the analytes in preparation for ionization using an electrospray ionization source. However, unlike traditional mass spectrometry (MS) techniques, it operates under ambient pressure and requires no sample preparation, thereby making it ideal for rapid sampling of thicker tissue sections for electrophysiological and other neuroscientific research studies. Studies interrogating neural synapses, or a specific neural circuit, typically employ thick, ex vivo tissue sections maintained under near-physiological conditions to preserve tissue viability and maintain the neural networks. Deep brain stimulation (DBS) is a surgical procedure used to treat the neurological symptoms that are associated with certain neurodegenerative and neuropsychiatric diseases. Parkinson's disease (PD) is a neurological disorder which is commonly treated with DBS therapy. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta portion of the brain. Here, we demonstrate that the LMJ-SS methodology can provide a platform for ex vivo analysis of the brain during electrical stimulation, such as DBS. We employ LMJ-SS in the ex vivo analysis of mouse brain tissue for monitoring dopamine during electrical stimulation of the striatum region. The mouse brain tissue was sectioned fresh post sacrifice and maintained in artificial cerebrospinal fluid to create near-physiological conditions before direct sampling using LMJ-SS. A selection of metabolites, including time-sensitive metabolites involved in energy regulation in the brain, were identified using standards, and the mass spectral database mzCloud was used to assess the feasibility of the methodology. Thereafter, the intensity of m/z 154 corresponding to protonated dopamine was monitored before

Ex vivo bupivacaine treatment results in increased adipogenesis of skeletal muscle cells in the rat.

PubMed

Yamanouchi, Keitaro; Nakamura, Katsuyuki; Takegahara, Yuki; Nakano, Shin-ichi; Nishihara, Masugi

2013-11-01

Intramuscular adipose tissue (IMAT) is observed in some skeletal muscle pathologies. IMAT is implicated not only in the disorders of muscle contraction, but also of metabolism and insulin sensitivity due to its nature as a secretary organ. Several studies indicate the presence of cells with adipogenic potential in skeletal muscle. However, the mechanism of fate specification that triggers these cells to enter an adipogenic program in vivo remains to be solved. In the present study, we examined whether activation of the adipogenic program of muscle-resident cells precedes their proliferation upon muscle injury. For this purpose, muscle injury was induced by injecting bupivacaine (BPVC) to excised skeletal muscle ex vivo. Cells isolated from ex vivo BPVC-treated muscle exhibited higher adipogenic potential than those from saline-treated muscle. Pre-plating exposure of skeletal muscle cells to basic fibroblast growth factor (bFGF) mimicked the effect of ex vivo BPVC-treatment, suggesting that bFGF released from extracellular matrix in response to muscle injury activates their adipogenic program. Interestingly, the number of myotubes were significantly reduced in the culture from BPVC-treated muscle, suggesting that adipocytes negatively regulate myogenesis. © 2013 Japanese Society of Animal Science.

Freesurfer cortical normative data for adults using Desikan-Killiany-Tourville and ex vivo protocols.

PubMed

Potvin, Olivier; Dieumegarde, Louis; Duchesne, Simon

2017-08-01

We recently built normative data for FreeSurfer morphometric estimates of cortical regions using its default atlas parcellation (Desikan-Killiany or DK) according to individual and scanner characteristics. We aimed to produced similar normative values for Desikan-Killianny-Tourville (DKT) and ex vivo-based labeling protocols, as well as examine the differences between these three atlases. Surfaces, thicknesses, and volumes of cortical regions were produced using cross-sectional magnetic resonance scans from the same 2713 healthy individuals aged 18-94 years as used in the reported DK norms. Models predicting regional cortical estimates of each hemisphere were produced using age, sex, estimated intracranial volume (eTIV), scanner manufacturer and magnetic field strength (MFS) as predictors. The DKT and DK models generally included the same predictors and produced similar R 2 . Comparison between DK, DKT, ex vivo atlases normative cortical measures showed that the three protocols generally produced similar normative values. Copyright © 2017 Elsevier Inc. All rights reserved.

Tumor associated antigen specific T-cell populations identified in ex vivo expanded TIL cultures.

PubMed

Junker, Niels; Kvistborg, Pia; Køllgaard, Tania; Straten, Per thor; Andersen, Mads Hald; Svane, Inge Marie

2012-01-01

Ex vivo expanded tumor infiltrating lymphocytes (TILs) from malignant melanoma (MM) and head & neck squamous cell carcinoma (HNSCC) share a similar oligoclonal composition of T effector memory cells, with HLA class I restricted lysis of tumor cell lines. In this study we show that ex vivo expanded TILs from MM and HNSCC demonstrate a heterogeneous composition in frequency and magnitude of tumor associated antigen specific populations by Elispot IFNγ quantitation. TILs from MM and HNSCC shared reactivity towards NY ESO-1, cyclin B1 and Bcl-x derived peptides. Additionally we show that dominating T-cell clones and functionality persists through out expansion among an oligoclonal composition of T-cells. Our findings mirror prior results on the oligoclonal composition of TIL cultures, further indicating a potential for a broader repertoire of specific effector cells recognizing the heterogeneous tumors upon adoptive transfer; increasing the probability of tumor control by minimizing immune evasion by tumor cell escape variants. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

PubMed Central

Rohban, Rokhsareh; Reinisch, Andreas; Etchart, Nathalie; Schallmoser, Katharina; Hofmann, Nicole A.; Szoke, Krisztina; Brinchmann, Jan E.; Rad, Ehsan Bonyadi; Rohde, Eva; Strunk, Dirk

2013-01-01

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo. PMID:23826172

Release and in vitro skin permeation of polyphenols from cosmetic emulsions.

PubMed

Zillich, O V; Schweiggert-Weisz, U; Hasenkopf, K; Eisner, P; Kerscher, M

2013-10-01

Polyphenols are natural antioxidants, which can inhibit oxidative chain reactions in human skin and prevent therefore some skin diseases and premature ageing. A prerequisite of this behaviour is their permeation through the skin barrier, in particular the stratum corneum (SC). In this study, we investigated the skin permeation kinetic of polyphenols, incorporated to semisolid emulsions, and the release of polyphenols from the emulsions. Mixtures of model substances, consisting of catechin, epigallocatechin gallate (EGCG), resveratrol, quercetin, rutin and protocatechuic acid (PCA), were formulated into o/w emulsions with different oil phase content. The in vitro experiments were carried out in Franz-type diffusion cells by means of ex vivo pig skin and a cellulose membrane. The increased oil content in the emulsion led to a significant decrease in initial release coefficients (K(r)), diffusion coefficients within the formulation (D(v)) and skin permeation coefficients (K(p)), respectively. The study considered the dependence of K(r) on molecular weight and lipophilicity of polyphenolics. For both more hydrophilic and more lipophilic substance groups, the values for K(r) were inverse proportional to molecular weight. For catechin, quercetin, rutin, resveratrol and PCA, a good correlation between K(p) and K(r) parameters was obtained. The most permeable substance was PCA (K(p) = 1.2·10(-3) cm h(-1)), and the least permeable was quercetin (K(p) = 1.5·10(-5) cm h(-1)). All substances could pass the SC barrier and were found mostly in the epidermis and dermis, confirming the potential of polyphenols as anti-ageing active cosmetic ingredients. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

PubMed

Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

2016-01-01

Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. Copyright © 2015 Pava et al.

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

Soluble TNF receptor 1-secreting ex vivo-derived dendritic cells reduce injury after stroke.

PubMed

Works, Melissa G; Koenig, Jenny B; Sapolsky, Robert M

2013-09-01

Inflammation is a major factor in the progression of damage after stroke and in the clinic, current therapies treat the clot, not the resulting damage. We have developed a novel method of protein delivery that exploits the migration ability of leukocytes after ischemic stroke (transient middle cerebral artery occlusion; tMCAO). In our studies, ex vivo-derived dendritic cells (exDCs) migrate to the inflamed rat brain soon after tMCAO onset and the number of cells that remain in the brain after injection is significantly correlated with the amount of local inflammation at the injury site. In addition, exDCs transduced to overexpress soluble tumor necrosis factor (TNF) receptor1 (sTNFR1) produce functional cargo that is secreted and that blocks TNF-α bioavailability in vitro. When delivered at 6 hours post-tMCAO reperfusion, sTNFR1-exDC-treated rats show significantly smaller infarct size and decreased inflammation compared with animals treated with exDCs transduced with GFP lentivirus. These studies indicate that cell-mediated delivery of proteins may be a promising new approach to reduce brain damage after acute neurologic insult.

Comparison of In-Vitro and Ex-Vivo Wound Healing Assays for the Investigation of Diabetic Wound Healing and Demonstration of a Beneficial Effect of a Triterpene Extract

PubMed Central

Ueck, Christopher; Volksdorf, Thomas; Houdek, Pia; Vidal-y-Sy, Sabine; Sehner, Susanne; Ellinger, Bernhard; Lobmann, Ralf; Larena-Avellaneda, Axel; Reinshagen, Konrad; Ridderbusch, Ina; Kohrmeyer, Klaas; Moll, Ingrid; Daniels, Rolf; Werner, Philipp; Merfort, Irmgard; Brandner, Johanna M.

2017-01-01

Diabetes mellitus is a frequent cause for chronic, difficult-to-treat wounds. New therapies for diabetic wounds are urgently needed and in-vitro or ex-vivo test systems are essential for the initial identification of new active molecules. The aim of this study is to compare in-vitro and ex-vivo test systems for their usability for early drug screening and to investigate the efficacy of a birch bark triterpene extract (TE) that has been proven ex-vivo and clinically to accelerate non-diabetic wound healing (WH), in a diabetic context. We investigated in-vitro models for diabetic WH, i.e. scratch assays with human keratinocytes from diabetic donors or cultured under hyperglycaemic conditions and a newly developed porcine ex-vivo hyperglycaemic WH model for their potential to mimic delayed diabetic WH and for the influence of TE in these test systems. We show that keratinocytes from diabetic donors often fail to exhibit significantly delayed WH. For cells under hyperglycaemic conditions significant decrease is observed but is influenced by choice of medium and presence of supplements. Also, donor age plays a role. Interestingly, hyperglycaemic effects are mainly hyperosmolaric effects in scratch assays. Ex-vivo models under hyperglycaemic conditions show a clear and substantial decrease of WH, and here both glucose and hyperosmolarity effects are involved. Finally, we provide evidence that TE is also beneficial for ex-vivo hyperglycaemic WH, resulting in significantly increased length of regenerated epidermis to 188±16% and 183±11% (SEM; p<0.05) compared to controls when using two different TE formulations. In conclusion, our results suggest that microenvironmental influences are important in WH test systems and that therefore the more complex hyperglycaemic ex-vivo model is more suitable for early drug screening. Limitations of the in-vitro and ex-vivo models are discussed. Furthermore our data recommend TE as a promising candidate for in-vivo testings in diabetic

Real-Time Amperometric Recording of Extracellular H₂O₂ in the Brain of Immunocompromised Mice: An In Vitro, Ex Vivo and In Vivo Characterisation Study.

PubMed

Reid, Caroline H; Finnerty, Niall J

2017-07-08

We detail an extensive characterisation study on a previously described dual amperometric H₂O₂ biosensor consisting of H₂O₂ detection (blank) and degradation (catalase) electrodes. In vitro investigations demonstrated excellent H₂O₂ sensitivity and selectivity against the interferent, ascorbic acid. Ex vivo studies were performed to mimic physiological conditions prior to in vivo deployment. Exposure to brain tissue homogenate identified reliable sensitivity and selectivity recordings up to seven days for both blank and catalase electrodes. Furthermore, there was no compromise in pre- and post-implanted catalase electrode sensitivity in ex vivo mouse brain. In vivo investigations performed in anaesthetised mice confirmed the ability of the H₂O₂ biosensor to detect increases in amperometric current following locally perfused/infused H₂O₂ and antioxidant inhibitors mercaptosuccinic acid and sodium azide. Subsequent recordings in freely moving mice identified negligible effects of control saline and sodium ascorbate interference injections on amperometric H₂O₂ current. Furthermore, the stability of the amperometric current was confirmed over a five-day period and analysis of 24-h signal recordings identified the absence of diurnal variations in amperometric current. Collectively, these findings confirm the biosensor current responds in vivo to increasing exogenous and endogenous H₂O₂ and tentatively supports measurement of H₂O₂ dynamics in freely moving NOD SCID mice.

Molecular studies on di-sodium tartrate molecule

NASA Astrophysics Data System (ADS)

Divya, P.; Jayakumar, S.; George, Preethamary; Shubashree, N. S.; Ahmed. M, Anees

2015-06-01

Structural characterization is important for the development of new material. The acoustical parameters such as Free Length, Internal Pressure have been measured from ultrasonic velocity, density for di sodium tartrate an optically active molecule at different temperatures using ultrasonic interferometer of frequency (2MHZ). The ultrasonic velocity increases with increase in concentration there is an increase in solute-solvent interaction. The stability constant had been calculated. SEM with EDAX studies has been done for Di-sodium tartrate an optically active molecule.

Exploring sex differences in the adult zebra finch brain: In vivo diffusion tensor imaging and ex vivo super-resolution track density imaging.

PubMed

Hamaide, Julie; De Groof, Geert; Van Steenkiste, Gwendolyn; Jeurissen, Ben; Van Audekerke, Johan; Naeyaert, Maarten; Van Ruijssevelt, Lisbeth; Cornil, Charlotte; Sijbers, Jan; Verhoye, Marleen; Van der Linden, Annemie

2017-02-01

Zebra finches are an excellent model to study the process of vocal learning, a complex socially-learned tool of communication that forms the basis of spoken human language. So far, structural investigation of the zebra finch brain has been performed ex vivo using invasive methods such as histology. These methods are highly specific, however, they strongly interfere with performing whole-brain analyses and exclude longitudinal studies aimed at establishing causal correlations between neuroplastic events and specific behavioral performances. Therefore, the aim of the current study was to implement an in vivo Diffusion Tensor Imaging (DTI) protocol sensitive enough to detect structural sex differences in the adult zebra finch brain. Voxel-wise comparison of male and female DTI parameter maps shows clear differences in several components of the song control system (i.e. Area X surroundings, the high vocal center (HVC) and the lateral magnocellular nucleus of the anterior nidopallium (LMAN)), which corroborate previous findings and are in line with the clear behavioral difference as only males sing. Furthermore, to obtain additional insights into the 3-dimensional organization of the zebra finch brain and clarify findings obtained by the in vivo study, ex vivo DTI data of the male and female brain were acquired as well, using a recently established super-resolution reconstruction (SRR) imaging strategy. Interestingly, the SRR-DTI approach led to a marked reduction in acquisition time without interfering with the (spatial and angular) resolution and SNR which enabled to acquire a data set characterized by a 78μm isotropic resolution including 90 diffusion gradient directions within 44h of scanning time. Based on the reconstructed SRR-DTI maps, whole brain probabilistic Track Density Imaging (TDI) was performed for the purpose of super resolved track density imaging, further pushing the resolution up to 40μm isotropic. The DTI and TDI maps realized atlas

Integrity and stability of oral liposomes containing bile salts studied in simulated and ex vivo gastrointestinal media.

PubMed

Hu, Shunwen; Niu, Mengmeng; Hu, Fuqiang; Lu, Yi; Qi, Jianping; Yin, Zongning; Wu, Wei

2013-01-30

The objective of this study was to investigate the integrtity and stability of oral liposomes containing glycocholate (SGC-Lip) in simulated gastrointestinal (GI) media and ex vivo GI media from rats in comparison with conventional liposomes (CH-Lip) composed of soybean phosphatidylcholine and cholesterol. Membrane integrity of liposomes was evaluated by monitoring calcein release, particle size and distribution in different simulated GI media. The stability of liposomes encapsulating insulin was investigated in simulated GI fluids containing pepsin or pancreatin and ex vivo GI enzyme fluids. Simulated GI media with low pH or physiological bile salts resulted in significant increase in calcein release, but dynamic laser scattering data showed that the size and distribution were generally stable. SGC-Lip retained the major amount of the initially encapsulated insulin as compared with CH-Lip in simulated GI fluids (SGF, FaSSGF, SIF and FeSSIF-V2). SGC-Lip retained respectively 17.1% and 20.5% of the initially encapsulated insulin in ex vivo GI fluid, which were also significantly more than CH-Lip. These results suggested that SGC-Lip could protect insulin from degradation to some degree during their transit through the gastrointestinal tract and contributed to enhanced oral absorption. Copyright © 2012 Elsevier B.V. All rights reserved.

Alveolar macrophages from allergic lungs are not committed to a pro-allergic response and can reduce airway hyperresponsiveness following ex vivo culture

PubMed Central

Pouliot, P.; Spahr, A.; Careau, É.; Turmel, V.; Bissonnette, E. Y.

2016-01-01

Summary Background We already demonstrated that adoptive transfer of alveolar macrophages (AMs) from non-allergic rats into AM-depleted allergic rats prevents airway hyperresponsiveness (AHR). We also showed that AMs from non-sensitized, but not from sensitized, allergy-prone rats can prevent AHR following allergen challenge in sensitized allergic animals, establishing the importance of rat immunological status on the modulation of AM functions and suggesting that an allergic lung environment alters AM functions. Objective We investigated how the activation of allergic AMs can be modulated to reinstitute them with their capacity to reduce AHR. Methods AMs from sensitized Brown Norway rats were cultured ex vivo for up to 18 h in culture media to deprogram them from the influence of the allergic lung before being reintroduced into the lung of AM-depleted sensitized recipient. AHR and cytokines in bronchoalveolar lavage (BAL) were measured following allergen challenge. AMs stimulated ex vivo with Bacillus Calmette-Guerin(BCG) were used as positive controls as BCG induces a T-helper type 1 activation in AMs. Results AMs ex vivo cultured for 4–18 h reduced AHR to normal level. Interestingly, pro-allergic functions of AMs were dampened by 18 h culture and they reduced AHR even after spending 48 h in an allergic lung microenvironment. Furthermore, transfer of cultured AMs caused an increase in the levels of IFN-γ and IL-12 in BAL when compared with their ovalbumin control. After 18 h of ex vivo culture, AMs expressed reduced levels of TNF, IL-1α, IL-6, and Arginase-2 mRNAs compared with freshly isolated AMs, suggesting that ex vivo culture exempted AMs from lung stimuli that affected their functions. Conclusions There is a significant crosstalk between lung microenvironment and AMs, affecting their functions. It is also the first report showing that sensitized AMs can be modulated ex vivo to reduce lung pro-allergic environment, opening the way to therapies targetting

Single crystal growth and characterization of pure and sodium-modified copper tartrate

NASA Astrophysics Data System (ADS)

Quasim, I.; Firdous, A.; Want, B.; Khosa, S. K.; Kotru, P. N.

2008-12-01

Single crystal growth of pure and modified copper tartrate crystals bearing composition (Cu) x(Na) yC 4H 4O 6· nH 2O (where x=1, 0.77, 0.65; y=0, 0.23, 0.35) is achieved using gel technique. The optimum conditions required for the growth of these crystals are worked out. The morphological development of these crystals is studied using optical and scanning electron microscopy. The dominant habit faces of the grown copper tartrate crystals are (0 0 1) and (1 1 1). Calculation of the cell parameters using CRYSFIRE software suggests that the pure copper tartrate crystal belongs to orthorhombic system with space group P2 1/c whereas the modified copper tartrate falls under tetragonal system with the space group P4 2/nbc. The external morphological development is shown to remain unaffected in the modified copper tartrate. The stoichiometric composition of the crystals is established by EDAX analysis, CH analysis, FTIR spectroscopy and thermoanalytical techniques. Thermal analysis of the grown crystals suggests that pure copper tartrate is thermally stable up to 42.84 °C whereas the modified copper tartrate crystals are stable only up to 33.11 and 25.11 °C. Calculation of the percentage weight loss from the thermogram supplemented by EDAX/CH analysis and FTIR spectroscopy suggest that the chemical formula of pure copper tartrate crystal is CuC 4H 4O 6·3H 2O whereas the chemical formula for the modified copper tartrate crystals is (Cu) 0.77(Na) 0.23C 4H 4O 6·3H 2O and (Cu) 0.65(Na) 0.35 C 4H 4O 6·H 2O.

Chitosan-coated diacerein nanosuspensions as a platform for enhancing bioavailability and lowering side effects: preparation, characterization, and ex vivo/in vivo evaluation

PubMed Central

Allam, Ahmed N; Hamdallah, Sherif I; Abdallah, Ossama Y

2017-01-01

Nanodrug delivery systems have been widely reviewed for their use in several drug formulations to improve bioavailability, sustain effect, and decrease side effects of many candidate drugs. The objective of this study was to evaluate the potential of chitosan (CS)-coated nanosuspensions to enhance bioavailability and reduce the diarrheal side effect of diacerein (DCN) after oral administration. DCN nanosuspensions (DNS) were prepared by sonoprecipitation technique using different stabilizers at three different concentrations. The selected DNS with optimum particle size (PS), polydispersity index (PDI), and Zeta potential (ZP) was coated with three different concentrations of CS-coated DNS (CS-DNS) and screened. In vitro dissolution was performed for the selected lyophilized formulae and compared with DCN powder in addition to the assessment of drug crystallinity via scanning electron microscopy, X-ray powder diffraction, and differential scanning calorimetry. Ex vivo drug permeability using noneverted rat intestine, intraluminal content, and mucoadhesion evaluation was studied for nominated formulae in comparison to DCN suspension. Moreover, in vivo study, pharmacokinetic parameters, and evaluation of diarrheal potential were conducted after oral administration of selected formulae. Polyvinyl pyrrolidone (PVP)-stabilized DNS showed a significant increase (P≤0.05) in PS and PDI as the stabilizer concentration increased. PVP-stabilized DNS with the lowest CS concentration was protected from aggregation by lyophilization with mannitol. A remarked enhancement in dissolution parameters was observed in the nanocrystals’ formulae. Morphological examination and X-ray diffraction confirmed drug crystallinity. The intermediate permeation parameters of CS-DNS-F10, lowest rhein-to-DCN ratio in intraluminal content along with the highest percentage of mucoadhesive, could serve as a sustaining profile of coated formula. CS-DNS-F10 showed a significantly higher Cmax of 0.74�

Chitosan-coated diacerein nanosuspensions as a platform for enhancing bioavailability and lowering side effects: preparation, characterization, and ex vivo/in vivo evaluation.

PubMed

Allam, Ahmed N; Hamdallah, Sherif I; Abdallah, Ossama Y

2017-01-01

Nanodrug delivery systems have been widely reviewed for their use in several drug formulations to improve bioavailability, sustain effect, and decrease side effects of many candidate drugs. The objective of this study was to evaluate the potential of chitosan (CS)-coated nanosuspensions to enhance bioavailability and reduce the diarrheal side effect of diacerein (DCN) after oral administration. DCN nanosuspensions (DNS) were prepared by sonoprecipitation technique using different stabilizers at three different concentrations. The selected DNS with optimum particle size (PS), polydispersity index (PDI), and Zeta potential (ZP) was coated with three different concentrations of CS-coated DNS (CS-DNS) and screened. In vitro dissolution was performed for the selected lyophilized formulae and compared with DCN powder in addition to the assessment of drug crystallinity via scanning electron microscopy, X-ray powder diffraction, and differential scanning calorimetry. Ex vivo drug permeability using noneverted rat intestine, intraluminal content, and mucoadhesion evaluation was studied for nominated formulae in comparison to DCN suspension. Moreover, in vivo study, pharmacokinetic parameters, and evaluation of diarrheal potential were conducted after oral administration of selected formulae. Polyvinyl pyrrolidone (PVP)-stabilized DNS showed a significant increase ( P ≤0.05) in PS and PDI as the stabilizer concentration increased. PVP-stabilized DNS with the lowest CS concentration was protected from aggregation by lyophilization with mannitol. A remarked enhancement in dissolution parameters was observed in the nanocrystals' formulae. Morphological examination and X-ray diffraction confirmed drug crystallinity. The intermediate permeation parameters of CS-DNS-F10, lowest rhein-to-DCN ratio in intraluminal content along with the highest percentage of mucoadhesive, could serve as a sustaining profile of coated formula. CS-DNS-F10 showed a significantly higher C max of 0.74�

Comparison of the Pharmacokinetics and Ex Vivo Antimalarial Activities of Artesunate-Amodiaquine and Artemisinin-Piperaquine in Healthy Volunteers for Preselection Malaria Therapy.

PubMed

Quang, Nguyen Ngoc; Chavchich, Marina; Anh, Chu Xuan; Birrell, Geoffrey W; van Breda, Karin; Travers, Thomas; Rowcliffe, Kerryn; Edstein, Michael D

2018-05-07

The pharmacokinetics (PK) and ex vivo activity (pharmacodynamics [PD]) of two artemisinin combination therapies (ACTs) (artemisinin-piperaquine [ARN-PPQ] [Artequick ® ] and artesunate-amodiaquine [ARS-AQ] [Coarsucam ™ ]) in healthy Vietnamese volunteers were compared following 3-day courses of the ACTs for the preselection of the drugs for falciparum malaria therapy. For PK analysis, serial plasma samples were collected from two separate groups of 22 volunteers after ACT administration. Of these volunteers, ex vivo activity was assessed in plasma samples from seven volunteers who received both ACTs. The area under the concentration-time curve (AUC 0-∞ ) was 3.6-fold higher for dihydroartemisinin (active metabolite of ARS) than that for ARN, whereas the AUC 0-∞ of desethylamodiaquine (active metabolite of AQ) was 2.0-fold lower than that of PPQ. Based on the 50% inhibitory dilution values of the volunteers' plasma samples collected from 0.25 to 3 hours after the last dose, the ex vivo activity of ARS-AQ was 2.9- to 16.2-fold more potent than that of ARN-PPQ against the drug-sensitive D6 Plasmodium falciparum line. In addition, at 1.5, 4.0, and 24 hours after the last dose, the ex vivo activity of ARS-AQ was 20.8-, 3.5-, and 8.5-fold more potent than that of ARN-PPQ against the ARN-sensitive MRA1239 line. By contrast, at 1.5 hours, the ex vivo activity of ARS-AQ was 5.4-fold more active than that of ARN-PPQ but had similar activities at 4 and 24 hours against the ARN-resistant MRA1240 line. The PK-PD data suggest that ARS-AQ possesses superior antimalarial activity than that of ARN-PPQ and would be the preferred ACT for further in vivo efficacy testing in multidrug-resistant falciparum malaria areas.

Comprehensive Antiretroviral Restriction Factor Profiling Reveals the Evolutionary Imprint of the ex Vivo and in Vivo IFN-β Response in HTLV-1-Associated Neuroinflammation.

PubMed

Leal, Fabio E; Menezes, Soraya Maria; Costa, Emanuela A S; Brailey, Phillip M; Gama, Lucio; Segurado, Aluisio C; Kallas, Esper G; Nixon, Douglas F; Dierckx, Tim; Khouri, Ricardo; Vercauteren, Jurgen; Galvão-Castro, Bernardo; Saraiva Raposo, Rui Andre; Van Weyenbergh, Johan

2018-01-01

HTLV-1-Associated Myelopathy (HAM/TSP) is a progressive neuroinflammatory disorder for which no disease-modifying treatment exists. Modest clinical benefit from type I interferons (IFN-α/β) in HAM/TSP contrasts with its recently identified IFN-inducible gene signature. In addition, IFN-α treatment in vivo decreases proviral load and immune activation in HAM/TSP, whereas IFN-β therapy decreases tax mRNA and lymphoproliferation. We hypothesize this "IFN paradox" in HAM/TSP might be explained by both cell type- and gene-specific effects of type I IFN in HTLV-1-associated pathogenesis. Therefore, we analyzed ex vivo transcriptomes of CD4 + T cells, PBMCs and whole blood in healthy controls, HTLV-1-infected individuals, and HAM/TSP patients. First, we used a targeted approach, simultaneously quantifying HTLV-1 mRNA (HBZ, Tax), proviral load and 42 host genes with known antiretroviral (anti-HIV) activity in purified CD4 + T cells. This revealed two major clusters ("antiviral/protective" vs. "proviral/deleterious"), as evidenced by significant negative (TRIM5/TRIM22/BST2) vs. positive correlation (ISG15/PAF1/CDKN1A) with HTLV-1 viral markers and clinical status. Surprisingly, we found a significant inversion of antiretroviral activity of host restriction factors, as evidenced by opposite correlation to in vivo HIV-1 vs. HTLV-1 RNA levels. The anti-HTLV-1 effect of antiviral cluster genes was significantly correlated to their adaptive chimp/human evolution score, for both Tax mRNA and PVL. Six genes of the proposed antiviral cluster underwent lentivirus-driven purifying selection during primate evolution (TRIM5/TRIM22/BST2/APOBEC3F-G-H), underscoring the cross-retroviral evolutionary imprint. Secondly, we examined the genome-wide type I IFN response in HAM/TSP patients, following short-term ex vivo culture of PBMCs with either IFN-α or IFN-β. Microarray analysis evidenced 12 antiretroviral genes (including TRIM5α/TRIM22/BST2) were significantly up-regulated by IFN

Can we use an ex vivo continuous hemofiltration model to describe the adsorption and elimination of meropenem and piperacillin?

PubMed

Jamal, Janattul-Ain; Udy, Andrew A; Wallis, Steven C; Ranganathan, Dwarakanathan; McWhinney, Brett C; Ungerer, Jacobus P J; Lipman, Jeffrey; Roberts, Jason A

2015-08-01

To determine the adsorption and elimination characteristics of meropenem and piperacillin during simulated continuous renal replacement therapy (CRRT), and to compare the observed data from this ex vivo study with previous data from clinical studies. This was an experimental study utilizing a modified CRRT circuit and polysulfone membrane (1.2 m2), circulated with a blood-crystalloid mixture. Adsorption onto the CRRT circuit was tested over a 4-h period, and clearance was assessed separately using variable continuous hemofiltration settings. A rapid 9% reduction in circulating meropenem and piperacillin concentrations was observed at approximately 0.5 and 1.0 h for each antibiotic, respectively. The post-dilution setting was associated with a significantly higher sieving coefficient (Sc) and filter clearance (CLfilter) (mean ± SD) (Sc 1.14 ± 0.10 versus 1.06 ± 0.04; CLfilter 19.05 ± 1.63 versus 17.59 ± 0.62 ml/min, P values < 0.05) for meropenem. No significant differences were observed for piperacillin pharmacokinetics. Clinically comparable Sc data were observed between data obtained from the ex vivo study and data from previous clinical studies, for both antibiotics. Meropenem and piperacillin appear to be rapidly adsorbed into the CRRT circuit, and the delivery site of fluid replacement significantly influences meropenem pharmacokinetics. However, these findings are likely to be clinically insignificant and not affect dosing requirements. This ex vivo method could be a surrogate for future clinical pharmacokinetic studies of CRRT. Further research is required to explore the applicability of the ex vivo method to further characterize antibiotic pharmacokinetics during CRRT.

Instrumented urethral catheter and its ex vivo validation in a sheep urethra

NASA Astrophysics Data System (ADS)

Ahmadi, Mahdi; Rajamani, Rajesh; Timm, Gerald; Sezen, Serdar

2017-03-01

This paper designs and fabricates an instrumented catheter for instantaneous measurement of distributed urethral pressure profiles. Since the catheter enables a new type of urological measurement, a process for accurate ex vivo validation of the catheter is developed. A flexible sensor strip is first fabricated with nine pressure sensors and integrated electronic pads for an associated sensor IC chip. The flexible sensor strip and associated IC chip are assembled on a 7 Fr Foley catheter. A sheep bladder and urethra are extracted and used in an ex vivo set up for verification of the developed instrumented catheter. The bladder-urethra are suspended in a test rig and pressure cuffs placed to apply known static and dynamic pressures around the urethra. A significant challenge in the performance of the sensor system is the presence of parasitics that introduce large bias and drift errors in the capacitive sensor signals. An algorithm based on use of reference parasitic transducers is used to compensate for the parasitics. Extensive experimental results verify that the developed compensation method works effectively. Results on pressure variation profiles circumferentially around the urethra and longitudinally along the urethra are presented. The developed instrumented catheter will be useful in improved urodynamics to more accurately diagnose the source of urinary incontinence in patients.

In-vivo and ex-vivo spectrofluorometric and imaging study of liposome uptake by the liver using a pH-sensitive probe

NASA Astrophysics Data System (ADS)

Soulie-Begu, Sylvie; Devoisselle, Jean-Marie; Mordon, Serge R.

1995-04-01

Liposomes are known to be uptaken by the liver cells after intraveinous injection. Only few techniques are available to follow this process in vivo like nuclear magnetic resonance spectroscopy or scintigraphy. Intracellular pathway and liposomes localization in the different liver cells require sacrifice of the animals, cells separation and electronic microscopy, then little is known about liposomes kinetic uptake by the acidic intracellular compartments in vivo. We propose in this study a new method to follow liposomes uptake in the liver in vivo using a fluorescent pH sensitive probe 5,6-carboxyfluorescein and two different composition of liposomes: phospholipids DSPC/Chol and DMPC in order to evaluate the influence of the formulation on the release characteristics of liposomes in the lysosomes. We have already demonstrated the ability of the fluorescence spectroscopy and imaging using a pH dependent probe to monitor pH in living tissues. As pH of lysosomes is very low, the kinetic liposomes uptake in this intracellular acidic compartment is followed by monitoring the pH of the whole liver in vivo and ex vivo. Carboxyfluorescein is used at high concentration (100 mM) in order to quench its fluorescence. Liposomes are injected to Wistar rats into the penil vein. After laparotomy, fluorescence spectra and images are recorded during two hours. Results show a clear relationship between formulation of liposomes and stability in the acidic compartments of hepatic cells. After sacrifice and flush with cold saline solution, pH of the liver ex vivo is found to be 5.0-5.5. Data show a rapid clearance of release dye and an uptake of liposomes by the liver cells and, as liposomes penetrate in the acidic compartment, dye is released from liposomes and is delivered in lysosomes leading to the decrease of the pH.

Energy Dissipation in Ex-Vivo Porcine Liver during Electrosurgery

PubMed Central

Karaki, Wafaa; Akyildiz, Ali; De, Suvranu

2017-01-01

This paper explores energy dissipation in ex-vivo liver tissue during radiofrequency current excitation with application in electrosurgery. Tissue surface temperature for monopolar electrode configuration is measured using infrared thermometry. The experimental results are fitted to a finite element model for transient heat transfer taking into account energy storage and conduction in order to extract information about “apparent” specific heat, which encompasses storage and phase change. The average apparent specific heat determined for low temperatures is in agreement with published data. However, at temperatures approaching the boiling point of water, apparent specific heat increases by a factor of five, indicating that vaporization plays an important role in the energy dissipation through latent heat loss. PMID:27479955

Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential.

PubMed

McNiece, Ian K; Almeida-Porada, Graça; Shpall, Elizabeth J; Zanjani, Esmail

2002-06-01

Cord blood (CB) products are becoming routinely used in unrelated allogeneic transplantation for smaller pediatric patients. Because of the low numbers of cells in CB compared to bone marrow or peripheral blood progenitor cells, their use is more limited in larger adults. Therefore, we developed ex vivo expansion conditions for CB and currently are transplanting ex vivo expanded CB products to patients receiving high-dose chemotherapy. As there is concern that ex vivo expansion may exhaust long-term engrafting cells, the current clinical protocols consist of both an expanded fraction and an unexpanded fraction. To determine the effect of expansion culture on long-term engrafting cells, we evaluated the short- and long-term engrafting potential of ex vivo expanded CB using a fetal sheep xenogeneic transplant model. CD 34(+) cells were selected from CB products and cultured in a two-step procedure in the presence of stem cell factor, megakaryocyte growth and differentiation factor, and granulocyte colony-stimulating factor for 14 days. Starting cells (CD34(+) cells), and cultured cells (day 7 and day 14 cells) were transplanted in 60-day-old fetal sheep and evaluated at various time points post transplant for the presence of human cells. Long-term engrafting cells were assessed by serial passage into secondary and tertiary recipients. Day 14 expanded CB cells provided more rapid engraftment than either the day 7 expanded cells or the day 0 cells; however, this engraftment was transient, and no human cells were detectable at 16 months post transplant in the animals that received the day 14 expanded cells. Day 0 cells had engrafted animals at 2 months post transplant and both the day 0 and day 7 cells persisted to 16 months or longer. In the secondary animals, the day 0 and day 7 cells engrafted equivalently at 3 months post transplant; however, no secondary engraftment resulted from the day 14 cells. The levels of engraftment in secondary animals receiving day 7 cells

Transdermal permeation of geniposide in the herbal complex liniment in vivo and in vitro.

PubMed

Wang, Yugang; Li, Lele; Li, Huiying; Zhu, Zhaoyun; Hua, Lei; Lei, Fan; Kheir, Michael M; Du, Lijun

2010-06-15

Zhongtong Caji, a kind of liniment, is a traditional Chinese medicinal formula that is widely used for clinical treatment of inflammation and sprains. In this study, the principal effective compound of this formula, geniposide, was used as a criterion to represent the transdermal permeability of the whole formula. A passive diffusion of Zhongtong Caji through the stratum corneum was discovered by an in vitro experiment. The dosage-content relationship detected in subcutaneous tissue after in vivo drug administration was further evidence of its permeation. Blood analysis after different dosages showed that the geniposide could be absorbed and accumulated by subcutaneous tissue within 1h after drug administration, and it would be eliminated by blood circulation 1h after drug treatment. Copyright 2010 Elsevier B.V. All rights reserved.

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

PubMed

Zhdanov, A V; Golubeva, A V; Okkelman, I A; Cryan, J F; Papkovsky, D B

2015-10-01

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-μm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 μM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 μM across the cell, or ∼0.6 μM/μm. Deeply deoxygenated (5-15 μM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues. Copyright © 2015 the American Physiological Society.

Ex Vivo Expanded Human Regulatory T Cells Can Prolong Survival of a Human Islet Allograft in a Humanized Mouse Model

PubMed Central

Wu, Douglas C.; Hester, Joanna; Nadig, Satish N.; Zhang, Wei; Trzonkowski, Piotr; Gray, Derek; Hughes, Stephen; Johnson, Paul; Wood, Kathryn J.

2013-01-01

Background Human regulatory T cells (Treg) offer an attractive adjunctive therapy to reduce current reliance on lifelong, nonspecific immunosuppression after transplantation. Here, we evaluated the ability of ex vivo expanded human Treg to prevent the rejection of islets of Langerhans in a humanized mouse model and examined the mechanisms involved. Methods We engrafted human pancreatic islets of Langerhans into the renal subcapsular space of immunodeficient BALB/c.rag2−/−.cγ−/− mice, previously rendered diabetic via injection of the β-cell toxin streptozocin. After the establishment of stable euglycemia, mice were reconstituted with allogeneic human peripheral blood mononuclear cells (PBMC) and the resultant alloreactive response studied. Ex vivo expanded CD25highCD4+ human Treg, which expressed FoxP3, CTLA-4, and CD62L and remained CD127low, were then cotransferred together with human PBMC and islet allografts and monitored for evidence of rejection. Results Human islets transplanted into diabetic immunodeficient mice reversed diabetes but were rejected rapidly after the mice were reconstituted with allogeneic human PBMC. Cotransfer of purified, ex vivo expanded human Treg prolonged islet allograft survival resulting in the accumulation of Treg in the peripheral lymphoid tissue and suppression of proliferation and interferon-γ production by T cells. In vitro, Treg suppressed activation of signal transducers and activators of transcription and inhibited the effector differentiation of responder T cells. Conclusions Ex vivo expanded Treg retain regulatory activity in vivo, can protect a human islet allograft from rejection by suppressing signal transducers and activators of transcription activation and inhibiting T-cell differentiation, and have clinical potential as an adjunctive cellular therapy. PMID:23917725

Expansion of Human and Murine Hematopoietic Stem and Progenitor Cells Ex Vivo without Genetic Modification Using MYC and Bcl-2 Fusion Proteins

PubMed Central

Bird, Gregory A.; Polsky, Avital; Estes, Patricia; Hanlon, Teri; Hamilton, Haley; Morton, John J.; Gutman, Jonathan; Jimeno, Antonio

2014-01-01

The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use. PMID:25170611

In vitro and ex vivo microbial leakage assessment in endodontics: A literature review.

PubMed

Savadkouhi, Sohrab Tour; Bakhtiar, Hengameh; Ardestani, Safoura Emami

2016-01-01

The aim of this study was to perform a literature review of published in-vitro and ex-vivo studies, which evaluated microbial leakage in endodontics in the past 10 years. A comprehensive electronic literature search was carried out in PubMed database for English articles published from 2005 to 2016 using the keywords "endodontics," " in vitro ," " ex vivo ," "microbial leakage," "microbial penetration," "saliva," " Enterococcus faecalis ," " E. faecalis ," "endodontic sealers," "temporary filling material," "apical plug," "mineral trioxide aggregate," and "MTA." The keywords were combined using Boolean operators AND/OR. Based on our search strategy, 33 relevant articles were included in the study. There are three main methods for assessment of bacterial microleakage, namely, (A) the dual-chamber leakage model, (B) detection of bacteria using a scanning electron microscope (SEM), and (C) polymerase chain reaction. All bacterial leakage models have some limitations and may yield different results compared to other microleakage evaluation techniques (i.e., dye penetration, fluid filtration, or electrochemical tests). The results of SEM correlated with those of microbial leakage test in most studies. Microbial leakage test using saliva better simulates the clinical setting for assessment of the leakage of single or mixed bacterial species.

Ex vivo mouse brain microscopy at 15T with loop-gap RF coil.

PubMed

Cohen, Ouri; Ackerman, Jerome L

2018-04-18

The design of a loop-gap-resonator RF coil optimized for ex vivo mouse brain microscopy at ultra high fields is described and its properties characterized using simulations, phantoms and experimental scans of mouse brains fixed in 10% formalin containing 4 mM Magnevist™. The RF (B 1 ) and magnetic field (B 0 ) homogeneities are experimentally quantified and compared to electromagnetic simulations of the coil. The coil's performance is also compared to a similarly sized surface coil and found to yield double the sensitivity. A three-dimensional gradient-echo (GRE) sequence is used to acquire high resolution mouse brain scans at (47 μm) 3 resolution in 1.8 h and a 20 × 20 × 19 μm 3 resolution in 27 h. The high resolution obtained permitted clear visualization and identification of multiple structures in the ex vivo mouse brain and represents, to our knowledge, the highest resolution ever achieved for a whole mouse brain. Importantly, the coil design is simple and easy to construct. Copyright © 2018 Elsevier Inc. All rights reserved.

Synthetic aperture ultrasound imaging with a ring transducer array: preliminary ex vivo results.

PubMed

Qu, Xiaolei; Azuma, Takashi; Yogi, Takeshi; Azuma, Shiho; Takeuchi, Hideki; Tamano, Satoshi; Takagi, Shu

2016-10-01

The conventional medical ultrasound imaging has a low lateral spatial resolution, and the image quality depends on the depth of the imaging location. To overcome these problems, this study presents a synthetic aperture (SA) ultrasound imaging method using a ring transducer array. An experimental ring transducer array imaging system was constructed. The array was composed of 2048 transducer elements, and had a diameter of 200 mm and an inter-element pitch of 0.325 mm. The imaging object was placed in the center of the ring transducer array, which was immersed in water. SA ultrasound imaging was then employed to scan the object and reconstruct the reflection image. Both wire phantom and ex vivo experiments were conducted. The proposed method was found to be capable of producing isotropic high-resolution images of the wire phantom. In addition, preliminary ex vivo experiments using porcine organs demonstrated the ability of the method to reconstruct high-quality images without any depth dependence. The proposed ring transducer array and SA ultrasound imaging method were shown to be capable of producing isotropic high-resolution images whose quality was independent of depth.

Effects of Acutely Elevated Hydrostatic Pressure in a Rat Ex Vivo Retinal Preparation

PubMed Central

Yoshitomi, Takeshi; Zorumski, Charles F.; Izumi, Yukitoshi

2010-01-01

Purpose. A new experimental glaucoma model was developed by using an ex vivo rat retinal preparation to examine the effects of elevated hydrostatic pressure on retinal morphology and glutamine synthetase (GS) activity. Methods. Ex vivo rat retinas were exposed to elevated hydrostatic pressure for 24 hours in the presence of glutamate or glutamate receptor antagonists and examined histologically. GS activity was assessed by colorimetric assay. Results. Pressure elevation induced axonal swelling in the nerve fiber layer. Axonal swelling was prevented by a combination of non-N-methyl-d-aspartate (non-NMDA) receptor antagonist and an NMDA receptor antagonist, indicating that the damage results from activation of both types of glutamate receptor. When glial function was preserved, the typical changes induced by glutamate consisted of reversible Müller cell swelling resulting from excessive glial glutamate uptake. The irreversible Müller cell swelling in hyperbaric conditions may indicate that pressure disrupts glutamate metabolism. Indeed, elevated pressure inhibited GS activity. In addition, glutamate exposure after termination of pressure exposure exhibited apparent Müller cell swelling. Conclusions. These results suggest that the neural degeneration observed during pressure elevation is caused by impaired glial glutamate metabolism after uptake. PMID:20688725

Dynamic impact force and association with structural damage to the knee joint: an ex-vivo study.

PubMed

Brill, Richard; Wohlgemuth, Walther A; Hempfling, Harald; Bohndorf, Klaus; Becker, Ursula; Welsch, Ulrich; Kamp, Alexander; Roemer, Frank W

2014-12-01

No systematic, histologically confirmed data are available concerning the association between magnitude of direct dynamic impact caused by vertical impact trauma and the resulting injury to cartilage and subchondral bone. The aim of this study was to investigate the association between dynamic impact and the resulting patterns of osteochondral injury in an ex-vivo model. A mechanical apparatus was employed to perform ex-vivo controlled dynamic vertical impact experiments in 110 pig knees with the femur positioned in a holding fixture. A falling body with a thrust plate and photo sensor was applied. The direct impact to the trochlear articular surface was registered and the resulting osteochondral injuries macroscopically and histologically correlated and categorized. The relationship between magnitude of direct impact and injury severity could be classified as stage I injuries (impact <7.3MPa): elastic deformation, no histological injury; stage II injuries (impact 7.3-9.6MPa): viscoelastic imprint of the cartilaginous surface, subchondral microfractures; stage III injuries (impact 9.6-12.7MPa): disrupted cartilage surface, chondral fissures and subchondral microfractures; stage IV injuries (impact >12.7MPa): osteochondral impression, histologically imprint and osteochondral macrofractures. The impact ranges and histologic injury stages determined from this vertical dynamic impact experiment allowed for a biomechanical classification of direct, acute osteochondral injury. In contrast to static load commonly applied in ex-vivo experiments, dynamic impact more realistically represents actual trauma to the knee joint.

US-guided application of Nd:YAG laser in porcine pancreatic tissue: an ex vivo study and numerical simulation.

PubMed

Di Matteo, Francesco; Martino, Margareth; Rea, Roberta; Pandolfi, Monica; Panzera, Francesco; Stigliano, Egidio; Schena, Emiliano; Saccomandi, Paola; Silvestri, Sergio; Pacella, Claudio Maurizio; Breschi, Luca; Perrone, Giuseppe; Coppola, Roberto; Costamagna, Guido

2013-11-01

Laser ablation (LA) with a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser is a minimally invasive approach able to achieve a high rate of complete tissue necrosis. In a previous study we described the feasibility of EUS-guided Nd:YAG pancreas LA performed in vivo in a porcine model. To establish the best laser setting of Nd:YAG lasers for pancreatic tissue ablation. A secondary aim was to investigate the prediction capability of a mathematical model on ablation volume. Ex vivo animal study. Hospital animal laboratory. Explanted pancreatic glands from 60 healthy farm pigs. Laser output powers (OP) of 1.5, 3, 6, 10, 15, and 20 W were supplied. Ten trials for each OP were performed under US guidance on ex vivo healthy porcine pancreatic tissue. Ablation volume (Va) and central carbonization volume (Vc) were measured on histologic specimens as the sum of the lesion areas multiplied by the thickness of each slide. The theoretical model of the laser-tissue interaction was based on the Pennes equation. A circumscribed ablation zone was observed in all histologic specimens. Va values grow with the increase of the OP up to 10 W and reach a plateau between 10 and 20 W. The trend of Vc values rises constantly until 20 W. The theoretical model shows a good agreement with experimental Va and Vc for OP between 1.5 and 10 W. Ex vivo study. Volumes recorded suggest that the best laser OP could be the lowest one to obtain similar Va with smaller Vc in order to avoid the risk of thermal injury to the surrounding tissue. The good agreement between the two models demonstrates the prediction capability of the theoretical model on laser-induced ablation volume in an ex vivo animal model and supports its potential use for estimating the ablation size at different laser OPs. Copyright © 2013 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.

The cryoablation of lung tissue using liquid nitrogen in gel and in the ex vivo pig lung.

PubMed

Nomori, Hiroaki; Yamazaki, Ikuo; Kondo, Toshiya; Kanno, Masaya

2017-02-01

To examine the efficiency of cryoablation using liquid nitrogen in lung tissue, we measured the size and temperature distribution of the frozen area (iceball) in gel and in the ex vivo pig lungs. Cryoprobes with diameters of 2.4 and 3.4 mm (2.4D and 3.4D, respectively) were used. Three temperature sensors were positioned at the surface of the cryoprobe and at distances of 0.5 and 1.5 cm from the cryoprobe. The ex vivo pig lungs were perfused with 37 °C saline and inflated using ventilator to simulate in vivo lung conditions. In gel, the 2.4D and 3.4D probes made iceballs of 3.9 ± 0.1 and 4.8 ± 0.3 cm in diameter, respectively, and the temperature at 1.5 cm from those probes reached -32 ± 8 and -53 ± 5 °C, respectively. In the pig lung, the 2.4D and 3.4D probes made iceballs of 5.2 ± 0.1 and 5.5 ± 0.4 cm in diameter, respectively, and the temperature at 1.5 cm from these probes reached -49 ± 5 and -58 ± 3 °C, respectively. Liquid nitrogen cryoablation using both 2.4D and 3.4D probes made iceballs that were of sufficient size, and effective temperatures were reached in both gel and the ex vivo pig lung.

Ex vivo expansion of alveolar macrophages with Mycobacterium tuberculosis from the resected lungs of patients with pulmonary tuberculosis

PubMed Central

Petrunina, Ekaterina; Umpeleva, Tatiana; Karskanova, Svetlana; Bayborodin, Sergey; Vakhrusheva, Diana; Kravchenko, Marionella; Skornyakov, Sergey

2018-01-01

Tuberculosis (TB), with the Mycobacterium tuberculosis (Mtb) as the causative agent, remains to be a serious world health problem. Traditional methods used for the study of Mtb in the lungs of TB patients do not provide information about the number and functional status of Mtb, especially if Mtb are located in alveolar macrophages. We have developed a technique to produce ex vivo cultures of cells from different parts of lung tissues surgically removed from patients with pulmonary TB and compared data on the number of cells with Mtb inferred by the proposed technique to the results of bacteriological and histological analyses used for examination of the resected lungs. The ex vivo cultures of cells obtained from the resected lungs of all patients were largely composed of CD14-positive alveolar macrophages, foamy or not, with or without Mtb. Lymphocytes, fibroblasts, neutrophils, and multinucleate Langhans giant cells were also observed. We found alveolar macrophages with Mtb in the ex vivo cultures of cells from the resected lungs of even those TB patients, whose sputum smears and lung tissues did not contain acid-fast Mtb or reveal growing Mtb colonies on dense medium. The detection of alveolar macrophages with Mtb in ex vivo culture as soon as 16–18 h after isolation of cells from the resected lungs of all TB patients suggests that the technique proposed for assessing the level of infection in alveolar macrophages of TB patients has higher sensitivity than do prolonged bacteriological or pathomorphological methods. The proposed technique allowed us to rapidly (in two days after surgery) determine the level of infection with Mtb in the cells of the resected lungs of TB patients and, by the presence or absence of Mtb colonies, including those with cording morphology, the functional status of the TB agent at the time of surgery. PMID:29401466

Ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration.

PubMed

Mort, Richard L; Keighren, Margaret; Hay, Leonard; Jackson, Ian J

2014-05-19

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles(1,2). Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture(3-6). Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue(3,6). High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture(6). By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.

Extradiscal ultrasound thermal therapy (ExDUSTT): evaluation in ex vivo and in vivo spine models (Invited Paper)

NASA Astrophysics Data System (ADS)

Diederich, Chris J.; Kinsey, Adam; Nau, William H.; Shu, Richard; Lotz, Jeffrey C.

2005-04-01

The application of heat to intervertebral discs is being clinically investigated for the treatment of discogenic back pain. The purpose of this study was to develop and test the feasibility of small ultrasound applicators that can be endoscopically placed adjacent to the disc, and deliver heating energy into the disc without puncturing the annular wall. Prototype devices were fabricated using curvilinear transducers (2.5-3.5 mm wide x 10 mm long, 5.4 - 6.5 MHz) that produce a narrow penetrating beam extending along the length of the ultrasound element. The transducer was affixed to either a flexible or rigid delivery catheter, and enclosed within an asymmetric coupling balloon with water-cooling flow. Bench measurements demonstrated 35-60% acoustic efficiencies, high-power output capabilities, and lightly focused beam patterns. The heating characteristics of these devices were evaluated with ex vivo and in vivo experiments within lumbar and cervical spine segments from sheep models and human cadaveric spine. The applicators were positioned adjacent to the annular wall of the surgically exposed discs. Ultrasound energy was focused directly into the disc to avoid heating the vertebral bodies. Multi-point thermocouple probes were placed throughout the disc to characterize the resultant temperature distributions. These studies demonstrated that ultrasound energy from these applicators penetrated the annular wall of the disc, and produced thermal coagulative temperatures of >60-65°C as far as 10 mm into the tissue. This study also showed that lower power levels and temperatures delivered for 10 minutes can generate a cytotoxic thermal dose of t43°C >240 min penetrating 5-10 mm from the annular wall.

Microultrasound characterisation of ex vivo porcine tissue for ultrasound capsule endoscopy

NASA Astrophysics Data System (ADS)

Lay, H. S.; Cox, B. F.; Sunoqrot, M.; Démoré, C. E. M.; Näthke, I.; Gomez, T.; Cochran, S.

2017-01-01

Gastrointestinal (GI) disease development and progression is often characterised by cellular and tissue architectural changes within the mucosa and sub-mucosa layers. Current clinical capsule endoscopy and other approaches are heavily reliant on optical techniques which cannot detect disease progression below the surface layer of the tissue. To enhance the ability of clinicians to detect cellular changes earlier and more confidently, both quantitative and qualitative microultrasound (μUS) techniques are investigated in healthy ex vivo porcine GI tissue. This work is based on the use of single-element, focussed μUS transducers made with micromoulded piezocomposite operating at around 48 MHz. To explore the possibility that μUS can detect Crohn’s disease and other inflammatory bowel diseases, ex vivo porcine small bowel tissue samples were cannulised and perfused with phosphate-buffered saline followed by various dilutions of polystyrene microspheres. Comparison with fluorescent imaging showed that the microspheres had infiltrated the microvasculature of the samples and that μUS was able to successfully detect this as a mimic of inflammation. Samples without microspheres were analysed using quantitative ultrasound to assess mechanical properties. Attenuation coefficients of 1.78 ± 0.66 dB/mm and 1.92 ± 0.77 dB/mm were obtained from reference samples which were surgically separated from the muscle layer. Six intact samples were segmented using a software algorithm and the acoustic impedance, Z, for varying tissue thicknesses, and backscattering coefficient, BSC, were calculated using the reference attenuation values and tabulated.

Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes.

PubMed

Isoler-Alcaraz, J; Fernández-Pérez, D; Larriba, E; Del Mazo, J

2017-10-18

Recently, an effective testis culture method using a gas-liquid interphase, capable of differentiate male germ cells from neonatal spermatogonia to spermatozoa has been developed. Nevertheless, this methodology needs deep analyses that allow future experimental approaches in basic, pathologic and/or reprotoxicologic studies. Because of this, we characterized at cellular and molecular levels the entire in vitro spermatogenic progression, in order to understand and evaluate the characteristics that define the spermatogenic process in ex vivo cultured testes compared to the in vivo development. Testicular explants of CD1 mice aged 6 and 10 days post-partum were respectively cultured during 55 and 89 days. Cytological and molecular approaches were performed, analyzing germ cell proportion at different time culture points, meiotic markers immunodetecting synaptonemal complex protein SYCP3 by immunocytochemistry and the relative expression of different marker genes along the differentiation process by Reverse Transcription - quantitative Polymerase Chain Reaction. In addition, microRNA and piwi-interactingRNA profiles were also evaluated by Next Generation Sequencing and bioinformatic approaches. The method promoted and maintained the spermatogenic process during 89 days. At a cytological level we detected spermatogenic development delays of cultured explants compared to the natural in vivo process. The expression of different spermatogenic stages gene markers correlated with the proportion of different cell types detected in the cytological preparations. In vitro progression analysis of the different spermatogenic cell types, from both 6.5 dpp and 10.5 dpp testes explants, has revealed a relative delay in relation to in vivo process. The expression of the genes studied as biomarkers correlates with the cytologically and functional detected progression and differential expression identified in vivo. After a first analysis of deep sequencing data it has been observed

Use of 1070 nm fiber lasers in oral surgery: preliminary ex vivo study with FBG temperature monitoring.

PubMed

Fornaini, Carlo; Merigo, Elisabetta; Poli, Federica; Cavatorta, Chiara; Rocca, Jean-Paul; Selleri, Stefano; Cucinotta, Annamaria

2017-12-31

The aim of this ex vivo study was to demonstrate the performances of 1070 nm fiber lasers for the ablation of oral tissues through the evaluation of the histological modifications made by a blind pathologist and the measurement of the thermal elevation during laser irradiation by a sensor based on a fiber Bragg grating. The source used was a pulsed fiber laser emitting at 1070 nm, with 20 W maximum average output power and 100 ns fixed pulse duration. Different tests were performed by changing the laser parameters, particularly the peak power of the pulses and the repetition rate. The tissue of the measurements demonstrated that the best properties in term of cutting capability and, at the same time, the lower thermal damages to the tissues can be obtained with a peak power of 3 kW, a repetition rate of 50 kHz and a speed of 5 mm/s. This ex vivo study showed that 1070 nm fiber lasers can be very useful in oral surgery, since they provide a reduced thermal elevation in the irradiated tissues, thus consequently respecting their biological structures. Moreover, this work demonstrates that FBG sensors, based on the optical fiber technology as the laser source considered for the tests, may be good instruments to record thermal elevation when applied to the ex vivo studies on animal models.

Evaluation of ex vivo produced endothelial progenitor cells for autologous transplantation in primates.

PubMed

Qin, Meng; Guan, Xin; Zhang, Yu; Shen, Bin; Liu, Fang; Zhang, Qingyu; Ma, Yupo; Jiang, Yongping

2018-01-22

Autologous transplantation of endothelial progenitor cells (EPCs) is a promising therapeutic approach in the treatment of various vascular diseases. We previously reported a two-step culture system for scalable generation of human EPCs derived from cord blood CD34 + cells ex vivo. Here, we now apply this culture system to expand and differentiate human and nonhuman primate EPCs from mobilized peripheral blood (PB) CD34 + cells for the therapeutic potential of autologous transplantation. The human and nonhuman primate EPCs from mobilized PB CD34 + cells were cultured according to our previously reported system. The generated adherent cells were then characterized by the morphology, surface markers, nitric oxide (NO)/endothelial NO synthase (eNOS) levels and Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake/fluorescein isothiocyanate (FITC)-lectin binding actives. Furthermore, the efficacy and safety studies were performed by autologous transplantation via hepatic portal vein injection in a nonhuman primate model with acute liver sinusoidal endothelial cell injury. The mobilized PB CD34 + cells from both human and nonhuman primate were efficiently expanded and differentiated. Over 2 × 10 8 adherent cells were generated from 20 mL mobilized primate PB (1.51 × 10 6  ± 3.39 × 10 5 CD34 + cells) by 36-day culture and more than 80% of the produced cells were identified as EPCs/endothelial cells (ECs). In the autologous transplant model, the injected EPC/ECs from nonhuman primate PB were scattered in the intercellular spaces of hepatocytes at the hepatic tissues 14 days post-transplantation, indicating successful migration and reconstitution in the liver structure as the functional EPCs/ECs. We successfully applied our previous two-step culture system for the generation of primate EPCs from mobilized PB CD34 + cells, evaluated the phenotypes ex vivo, and transplanted autologous EPCs/ECs in a nonhuman primate model. Our study indicates that

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT

EPA Science Inventory

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT.

S. R. Bielmeier1, A. S. Murr2, D. S. Best2, J. M. Goldman2, and M. G. Narotsky2

1 Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC, USA
2 Reproductive T...

Performance assessment of Pulse Wave Imaging using conventional ultrasound in canine aortas ex vivo and normal human arteries in vivo

PubMed Central

Li, Ronny X.; Qaqish, William; Konofagou, Elisa. E.

2015-01-01

The propagation behavior of the arterial pulse wave may provide valuable diagnostic information for cardiovascular pathology. Pulse Wave Imaging (PWI) is a noninvasive, ultrasound imaging-based technique capable of mapping multiple wall motion waveforms along a short arterial segment over a single cardiac cycle, allowing for the regional pulse wave velocity (PWV) and propagation uniformity to be evaluated. The purpose of this study was to improve the clinical utility of PWI using a conventional ultrasound system. The tradeoff between PWI spatial and temporal resolution was evaluated using an ex vivo canine aorta (n = 2) setup to assess the effects of varying image acquisition and signal processing parameters on the measurement of the PWV and the pulse wave propagation uniformity r2. PWI was also performed on the carotid arteries and abdominal aortas of 10 healthy volunteers (24.8 ± 3.3 y.o.) to determine the waveform tracking feature that would yield the most precise PWV measurements and highest r2 values in vivo. The ex vivo results indicated that the highest precision for measuring PWVs ~ 2.5 – 3.5 m/s was achieved using 24–48 scan lines within a 38 mm image plane width (i.e. 0.63 – 1.26 lines/mm). The in vivo results indicated that tracking the 50% upstroke of the waveform would consistently yield the most precise PWV measurements and minimize the error in the propagation uniformity measurement. Such findings may help establish the optimal image acquisition and signal processing parameters that may improve the reliability of PWI as a clinical measurement tool. PMID:26640603

Insight into k13-propeller gene polymorphism and ex vivo DHA-response profiles from Cameroonian isolates.

PubMed

Menard, Sandie; Tchoufack, Joëlle Njila; Maffo, Christelle Ngou; Nsango, Sandrine E; Iriart, Xavier; Abate, Luc; Tsapi, Majoline Tchioffo; Awono-Ambéné, Parfait H; Abega Mekongo, Francis A; Morlais, Isabelle; Berry, Antoine

2016-11-26

The spread of Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia is a major source of concern and the emergence of resistance in Africa would have dramatic consequences, by increasing malaria mortality and morbidity. It is therefore urgent to implement regular monitoring in sentinel sites in sub-Saharan Africa using robust and easy-to-implement tools. The prevalence of k13-propeller mutations and the phenotypic profiles are poorly known in sub-Saharan Africa. Here, the k13-propeller polymorphism was compared to both ex vivo susceptibility to DHA and early parasitological and clinical responses to artemisinin combination therapy (ACT). Plasmodium falciparum isolates were collected in 2015 in Yaoundé (Cameroon) from patients treated with dihydroartemisinin-piperaquine combination. Samples were analysed for their susceptibility to artemisinin using the k13-propeller sequencing, the ex vivo ring-stage survival assay, the in vivo parasite positive rate and the clinical statute at day 2. None of the collected isolates revealed the presence of resistance mutations in the k13-propeller sequence. The median ring-stage survival rate for all the 64 interpretable isolates after a 6-hour pulse of 700 nM dihydroartemisinin was low, 0.49% (IQR: 0-1.3). Total parasite clearance was observed for 87.5% of patients and the remaining parasitaemic isolates (12.5%) showed a high reduction of parasite load, ranging from 97.5 to 99.9%. Clinical symptoms disappeared in 92.8% of cases. This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon. Only synonymous mutations were found with a low prevalence (4.3%). A good association between k13 genotypes and the ex vivo ring-stage survival assay or parasitological and clinical data was obtained. These results give a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa.

TSPO activation modulates the effects of high pressure in a rat ex vivo glaucoma model

PubMed Central

Ishikawa, Makoto; Yoshitomi, Takeshi; Covey, Douglas F.; Zorumski, Charles F.; Izumi, Yukitoshi

2017-01-01

We previously reported that elevated pressure induces axonal swelling and facilitates the synthesis of the neurosteroid, allopregnanolone (AlloP), in the ex vivo rat retina. Exogenously applied AlloP attenuates the axonal swelling, suggesting that the neurosteroid plays a neuroprotective role against glaucomatous pressure-induced injuries, although mechanisms underlying neurosteroidogenesis have not been clarified. The aim of this study was to determine whether AlloP synthesis involves activation of translocator protein 18 kD (TSPO) and whether TSPO modulates pressure-induced retinal injury. Ex vivo rat retinas were exposed to various pressures (10, 35, or 75 mmHg) for 24 hours. Expression of TSPO, 5α-reductase (5aRD), and AlloP was examined by quantitative real-time RT-PCR, ELISA, immunohistochemistry, and LC-MS/MS. We also examined the effects of TSPO ligands on AlloP synthesis and retinal damage. In this acute model, quantitative real-time RT-PCR and ELISA analyses revealed that elevated pressure facilitated TSPO expression. Similarly, these methods also detected enhanced 5aRD (mostly type II), which was observed in retinal ganglion cells (RGC) and the inner nuclear layer (INL). Atriol, a TSPO antagonist, suppressed pressure mediated AlloP synthesis and induced more severe histological changes in the inner retina when combined with elevated pressure. PK11195, a TSPO ligand that facilitates AlloP synthesis by itself, remarkably diminished pressure-mediated retinal degeneration. These results suggest that AlloP synthesis is induced by sequential activation of TSPO and 5aRD in an ex vivo glaucoma model, and that TSPO agonists may serve as potential therapeutic agents for the prevention of pressure-induced retinal damage. PMID:27596950

Comprehensive Antiretroviral Restriction Factor Profiling Reveals the Evolutionary Imprint of the ex Vivo and in Vivo IFN-β Response in HTLV-1-Associated Neuroinflammation

PubMed Central

Leal, Fabio E.; Menezes, Soraya Maria; Costa, Emanuela A. S.; Brailey, Phillip M.; Gama, Lucio; Segurado, Aluisio C.; Kallas, Esper G.; Nixon, Douglas F.; Dierckx, Tim; Khouri, Ricardo; Vercauteren, Jurgen; Galvão-Castro, Bernardo; Saraiva Raposo, Rui Andre; Van Weyenbergh, Johan

2018-01-01

HTLV-1-Associated Myelopathy (HAM/TSP) is a progressive neuroinflammatory disorder for which no disease-modifying treatment exists. Modest clinical benefit from type I interferons (IFN-α/β) in HAM/TSP contrasts with its recently identified IFN-inducible gene signature. In addition, IFN-α treatment in vivo decreases proviral load and immune activation in HAM/TSP, whereas IFN-β therapy decreases tax mRNA and lymphoproliferation. We hypothesize this “IFN paradox” in HAM/TSP might be explained by both cell type- and gene-specific effects of type I IFN in HTLV-1-associated pathogenesis. Therefore, we analyzed ex vivo transcriptomes of CD4+ T cells, PBMCs and whole blood in healthy controls, HTLV-1-infected individuals, and HAM/TSP patients. First, we used a targeted approach, simultaneously quantifying HTLV-1 mRNA (HBZ, Tax), proviral load and 42 host genes with known antiretroviral (anti-HIV) activity in purified CD4+ T cells. This revealed two major clusters (“antiviral/protective” vs. “proviral/deleterious”), as evidenced by significant negative (TRIM5/TRIM22/BST2) vs. positive correlation (ISG15/PAF1/CDKN1A) with HTLV-1 viral markers and clinical status. Surprisingly, we found a significant inversion of antiretroviral activity of host restriction factors, as evidenced by opposite correlation to in vivo HIV-1 vs. HTLV-1 RNA levels. The anti-HTLV-1 effect of antiviral cluster genes was significantly correlated to their adaptive chimp/human evolution score, for both Tax mRNA and PVL. Six genes of the proposed antiviral cluster underwent lentivirus-driven purifying selection during primate evolution (TRIM5/TRIM22/BST2/APOBEC3F-G-H), underscoring the cross-retroviral evolutionary imprint. Secondly, we examined the genome-wide type I IFN response in HAM/TSP patients, following short-term ex vivo culture of PBMCs with either IFN-α or IFN-β. Microarray analysis evidenced 12 antiretroviral genes (including TRIM5α/TRIM22/BST2) were significantly up

Scavenging and antioxidant properties of different grape cultivars against ionizing radiation-induced liver damage ex vivo.

PubMed

Singha, Indrani; Das, Subir Kumar

2016-04-01

Ionizing radiation (IR) has become an integral part of the modern medicine--both for diagnosis as well as therapy. However, normal tissues or even distant cells also suffer IR-induced free radical insult. It may be more damaging in longer term than direct radiation exposure. Antioxidants provide protection against IR-induced damage. Grapes are the richest source of antioxidants. Here, we assessed the scavenging properties of four grape (Vitis vinifera) cultivars, namely Flame seedless (Black), Kishmish chorni (Black with reddish brown), Red globe (Red) and Thompson seedless mutant (Green), and also evaluated their protective action against γ-radiation-induced oxidative stress in liver tissue ex vivo. The scavenging abilities of grape seeds [2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC₅₀ = 0.008 ± 0.001 mg/mL), hydrogen peroxide (IC₅₀ = 0.49 to 0.8 mg/mL), hydroxyl radicals (IC₅₀ = 0.08 ± 0.008 mg/mL), and nitric oxide (IC₅₀ = 0.8 ± 0.08 mg/mL)] were higher than that of skin or pulp. Gamma (γ) radiation exposure to sliced liver tissues ex vivo from goat, @ 6 Gy significantly (P < 0.001) decreased reduced glutathione (GSH) content by 21.2% and also activities of catalase, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione s-transferase (GST) by 49.5, 66.0, 70.3, 73.6%, respectively. However, it increased thiobarbituric acid reactive substances (TBARS) by 2.04-fold and nitric oxide level by 48.6% compared to untreated group. Further increase in doses (10 or 16 Gy) of γ-radiation correspondingly decreased GSH content and enzyme activities, and increased TBARS and nitric oxide levels. Grape extract treatment prior to ionizing radiation exposure ameliorated theses effects at varying extent. The seed extracts exhibited strong antioxidant potential compared to skin or pulp extracts of different grape cultivars against oxidative damage by ionizing radiation (6 Gy, 10 Gy and 16 Gy) in sliced liver tissues ex vivo. Grape extracts at

Cochlear implants and ex vivo BDNF gene therapy protect spiral ganglion neurons.

PubMed

Rejali, Darius; Lee, Valerie A; Abrashkin, Karen A; Humayun, Nousheen; Swiderski, Donald L; Raphael, Yehoash

2007-06-01

Spiral ganglion neurons often degenerate in the deaf ear, compromising the function of cochlear implants. Cochlear implant function can be improved by good preservation of the spiral ganglion neurons, which are the target of electrical stimulation by the implant. Brain derived neurotrophic factor (BDNF) has previously been shown to enhance spiral ganglion survival in experimentally deafened ears. Providing enhanced levels of BDNF in human ears may be accomplished by one of several different methods. The goal of these experiments was to test a modified design of the cochlear implant electrode that includes a coating of fibroblast cells transduced by a viral vector with a BDNF gene insert. To accomplish this type of ex vivo gene transfer, we transduced guinea pig fibroblasts with an adenovirus with a BDNF gene cassette insert, and determined that these cells secreted BDNF. We then attached BDNF-secreting cells to the cochlear implant electrode via an agarose gel, and implanted the electrode in the scala tympani. We determined that the BDNF expressing electrodes were able to preserve significantly more spiral ganglion neurons in the basal turns of the cochlea after 48 days of implantation when compared to control electrodes. This protective effect decreased in the higher cochlear turns. The data demonstrate the feasibility of combining cochlear implant therapy with ex vivo gene transfer for enhancing spiral ganglion neuron survival.

Calcium Tartrate Tetrahydrate, Case Report of a Novel Human Kidney Stone.

PubMed

Kleinguetl, Colin; Williams, James C; Ibrahim, Samar A; Daudon, Michel; Bird, Erin T; El Tayeb, Marawan M

2017-01-01

Background: Calcium tartrate tetrahydrate has been reported as the main mineral in urinary stones in rats that have significant tartrate in their diet, but in humans, there has been only one mention of calcium tartrate stones in the form of bladder stone, and that case was in Africa. Case Presentation: Patient is a 34-year-old Caucasian male who presented with typical symptoms of nephrolithiasis. CT abd/pelvis (renal stone protocol) revealed a 2 cm nonobstructing stone of the right renal pelvis. Patient underwent an uncomplicated right percutaneous nephrolithotomy and was noted to be stone free after surgery. Stone analysis was difficult with regard to determining composition, but was finally identified as calcium tartrate tetrahydrate. Conclusion: This was an unusual case, as this is the first recorded case of a calcium tartrate tetrahydrate outside of Africa. This type of stone had only been mainly described in rat models with dl- bitartrate in their diet. Our patient was an otherwise healthy, relatively muscular individual with no obvious source for this stone other than a vitamin and amino acid supplement that he takes regularly that contains l-carnitine (as tartrate) and choline (as bitartrate and citrate). The prevalence of this stone type is presently unknown, as stone analysis laboratories have not had the ability to recognize it. Although a connection between the supplement and stone formation is conjecture at this time, we believe this necessitates further investigation.

Astaxanthin, a Carotenoid, Stimulates Immune Responses by Enhancing IFN-γ and IL-2 Secretion in Primary Cultured Lymphocytes in Vitro and ex Vivo.

PubMed

Lin, Kuan-Hung; Lin, Kao-Chang; Lu, Wan-Jung; Thomas, Philip-Aloysius; Jayakumar, Thanasekaran; Sheu, Joen-Rong

2015-12-29

Astaxanthin, a potent antioxidant carotenoid, plays a major role in modulating the immune response. In this study, we examined the immunomodulatory effects of astaxanthin on cytokine production in primary cultured lymphocytes both in vitro and ex vivo. Direct administration of astaxanthin (70-300 nM) did not produce cytotoxicity in lipopolysaccharide (LPS, 100 µg/ mL)- or concanavalin A (Con A, 10 µg/ mL)-activated lymphocytes, whereas astaxanthin alone at 300 nM induced proliferation of splenic lymphocytes (p < 0.05) in vitro. Although astaxanthin, alone or with Con A, had no apparent effect on interferon (INF-γ) and interleukin (IL-2) production in primary cultured lymphocytes, it enhanced LPS-induced INF-γ production. In an ex vivo experiment, oral administration of astaxanthin (0.28, 1.4 and 7 mg/kg/day) for 14 days did not cause alterations in the body or spleen weights of mice and also was not toxic to lymphocyte cells derived from the mice. Moreover, treatment with astaxanthin significantly increased LPS-induced lymphocyte proliferation ex vivo but not Con A-stimulated lymphocyte proliferation ex vivo. Enzyme linked immunosorbent assay (ELISA) analysis revealed that administration of astaxanthin significantly enhanced INF-γ production in response to both LPS and Con A stimulation, whereas IL-2 production increased only in response to Con A stimulation. Also, astaxanthin treatment alone significantly increased IL-2 production in lymphocytes derived from mice, but did not significantly change production of INF-γ. These findings suggest that astaxanthin modulates lymphocytic immune responses in vitro, and that it partly exerts its ex vivo immunomodulatory effects by increasing INF-γ and IL-2 production without inducing cytotoxicity.

Astaxanthin, a Carotenoid, Stimulates Immune Responses by Enhancing IFN-γ and IL-2 Secretion in Primary Cultured Lymphocytes in Vitro and ex Vivo

PubMed Central

Lin, Kuan-Hung; Lin, Kao-Chang; Lu, Wan-Jung; Thomas, Philip-Aloysius; Jayakumar, Thanasekaran; Sheu, Joen-Rong

2015-01-01

Astaxanthin, a potent antioxidant carotenoid, plays a major role in modulating the immune response. In this study, we examined the immunomodulatory effects of astaxanthin on cytokine production in primary cultured lymphocytes both in vitro and ex vivo. Direct administration of astaxanthin (70–300 nM) did not produce cytotoxicity in lipopolysaccharide (LPS, 100 µg/ mL)- or concanavalin A (Con A, 10 µg/ mL)-activated lymphocytes, whereas astaxanthin alone at 300 nM induced proliferation of splenic lymphocytes (p < 0.05) in vitro. Although astaxanthin, alone or with Con A, had no apparent effect on interferon (INF-γ) and interleukin (IL-2) production in primary cultured lymphocytes, it enhanced LPS-induced INF-γ production. In an ex vivo experiment, oral administration of astaxanthin (0.28, 1.4 and 7 mg/kg/day) for 14 days did not cause alterations in the body or spleen weights of mice and also was not toxic to lymphocyte cells derived from the mice. Moreover, treatment with astaxanthin significantly increased LPS-induced lymphocyte proliferation ex vivo but not Con A-stimulated lymphocyte proliferation ex vivo. Enzyme linked immunosorbent assay (ELISA) analysis revealed that administration of astaxanthin significantly enhanced INF-γ production in response to both LPS and Con A stimulation, whereas IL-2 production increased only in response to Con A stimulation. Also, astaxanthin treatment alone significantly increased IL-2 production in lymphocytes derived from mice, but did not significantly change production of INF-γ. These findings suggest that astaxanthin modulates lymphocytic immune responses in vitro, and that it partly exerts its ex vivo immunomodulatory effects by increasing INF-γ and IL-2 production without inducing cytotoxicity. PMID:26729100

Ex vivo and in vivo coherent Raman imaging of the peripheral and central nervous system

NASA Astrophysics Data System (ADS)

Huff, Terry Brandon

A hallmark of nervous system disorders is damage or degradation of the myelin sheath. Unraveling the mechanisms underlying myelin degeneration and repair represent one of the great challenges in medicine. This thesis work details the development and utilization of advanced optical imaging methods to gain insight into the structure and function of myelin in both healthy and diseased states in the in vivo environment. This first part of this thesis discusses ex vivo studies of the effects of high-frequency stimulation of spinal tissues on the structure of the node of Ranvier as investigated by coherent anti-Stokes Raman scattering (CARS) imaging (manuscript submitted to Journal of Neurosciece). Reversible paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation, beginning minutes after the onset and continuing for up to 10 min after stimulation was ceased. A mechanistic study revealed a Ca2+ dependent pathway: high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down. Also, the construction of dual-scanning CARS microscope for large area mapping of CNS tissues is detailed (Optics Express, 2008, 16:19396-193409). A confocal scanning head equipped with a rotating polygon mirror provides high speed, high resolution imaging and is coupled with a motorized sample stage to generate high-resolution large-area images of mouse brain coronal section and guinea pig spinal cord cross section. The polygon mirror decreases the mosaic acquisition time significantly without reducing the resolution of individual images. The ex vivo studies are then extended to in vivo imaging of mouse sciatic nerve tissue by CARS and second harmonic generation (SHG) imaging (Journal of Microscopy, 2007, 225: 175-182). Following a minimally invasive surgery to open the skin, CARS imaging of myelinated axons and SHG imaging of the

Effect of molecular structure of tartrates on chiral recognition of tartrate-boric acid complex chiral selectors in chiral microemulsion electrokinetic chromatography.

PubMed

Hu, Shao-Qiang; Chen, Yong-Lei; Zhu, Hua-Dong; Shi, Hai-Jun; Yan, Na; Chen, Xing-Guo

2010-08-20

Eight l-tartrates and a d-tartrate with different alcohol moieties were used as chiral oils to prepare chiral microemulsions, which were utilized in conjunction with borate buffer to separate the enantiomers of beta-blockers or structurally related compounds by the chiral microemulsion electrokinetic chromatography (MEEKC) method. Among them, six were found to have a relatively good chiral separation performance and their chiral recognition effect in terms of both enantioselectivity and resolution increases linearly with the number of carbon atoms in the alkyl group of alcohol moiety. The tartrates containing alkyl groups of different structures but the same number of carbon atoms, i.e. one of straight chain and one of branched chain, provide similar enantioseparations. The trend was elucidated according to the changes in the difference of the steric matching between the molecules of two enantiomers and chiral selector. Furthermore, it was demonstrated for the first time that a water insoluble solid compound, di-i-butyl l-tartrate (mp. 73.5 degrees C), can be used as an oil to prepare a stable microemulsion to be used in the chiral MEEKC successfully. And a critical effect of the microemulsion for chiral separation, which has never been reported before, was found in this experiment, namely providing a hydrophobic environment to strengthen the interactions between the chiral selector and enantiomers. Copyright 2010 Elsevier B.V. All rights reserved.

Vasomotor function in rat arteries after ex vivo and intragastric exposure to food-grade titanium dioxide and vegetable carbon particles.

PubMed

Jensen, Ditte Marie; Christophersen, Daniel Vest; Sheykhzade, Majid; Skovsted, Gry Freja; Lykkesfeldt, Jens; Münter, Rasmus; Roursgaard, Martin; Loft, Steffen; Møller, Peter

2018-02-26

Humans are continuously exposed to particles in the gastrointestinal tract. Exposure may occur directly through ingestion of particles via food or indirectly by removal of inhaled material from the airways by the mucociliary clearance system. We examined the effects of food-grade particle exposure on vasomotor function and systemic oxidative stress in an ex vivo study and intragastrically exposed rats. In an ex vivo study, aorta rings from naïve Sprague-Dawley rats were exposed for 30 min to food-grade TiO 2 (E171), benchmark TiO 2 (Aeroxide P25), food-grade vegetable carbon (E153) or benchmark carbon black (Printex 90). Subsequently, the vasomotor function was assessed in wire myographs. In an in vivo study, lean Zucker rats were exposed intragastrically once a week for 10 weeks to vehicle, E171 or E153. Doses were comparable to human daily intake. Vasomotor function in the coronary arteries and aorta was assessed using wire myographs. Tetrahydrobiopterin, ascorbate, malondialdehyde and asymmetric dimethylarginine were measured in blood as markers of oxidative stress and vascular function. Direct exposure of E171 to aorta rings ex vivo increased the acetylcholine-induced vasorelaxation and 5-hydroxytryptamine-induced vasocontraction. E153 only increased acetylcholine-induced vasorelaxation, and Printex 90 increased the 5-hydroxytryptamine-induced vasocontraction, whereas Aeroxide P25 did not affect the vasomotor function. In vivo exposure showed similar results as ex vivo exposure; increased acetylcholine-induced vasorelaxation in coronary artery segments of E153 and E171 exposed rats, whereas E171 exposure altered 5-hydroxytryptamine-induced vasocontraction in distal coronary artery segments. Plasma levels of markers of oxidative stress and vascular function showed no differences between groups. Gastrointestinal tract exposure to E171 and E153 was associated with modest albeit statistically significant alterations in the vasocontraction and vasorelaxation

Organotypic Cultures from the Adult CNS: A Novel Model to Study Demyelination and Remyelination Ex Vivo.

PubMed

Tan, Glaiza A; Furber, Kendra L; Thangaraj, Merlin P; Sobchishin, LaRhonda; Doucette, J Ronald; Nazarali, Adil J

2018-01-01

Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair.

Monitoring UV-induced signalling pathways in an ex vivo skin organ culture model using phospho-antibody array.

PubMed

Lenain, Christelle; Gamboa, Bastien; Perrin, Agnes; Séraïdaris, Alexia; Bertino, Béatrice; Rival, Yves; Bernardi, Mathieu; Piwnica, David; Méhul, Bruno

2018-05-01

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Elastic Cherenkov effects in transversely isotropic soft materials-II: Ex vivo and in vivo experiments

NASA Astrophysics Data System (ADS)

Li, Guo-Yang; He, Qiong; Qian, Lin-Xue; Geng, Huiying; Liu, Yanlin; Yang, Xue-Yi; Luo, Jianwen; Cao, Yanping

2016-09-01

In part I of this study, we investigated the elastic Cherenkov effect (ECE) in an incompressible transversely isotropic (TI) soft solid using a combined theoretical and computational approach, based on which an inverse method has been proposed to measure both the anisotropic and hyperelastic parameters of TI soft tissues. In this part, experiments were carried out to validate the inverse method and demonstrate its usefulness in practical measurements. We first performed ex vivo experiments on bovine skeletal muscles. Not only the shear moduli along and perpendicular to the direction of muscle fibers but also the elastic modulus EL and hyperelastic parameter c2 were determined. We next carried out tensile tests to determine EL, which was compared with the value obtained using the shear wave elastography method. Furthermore, we conducted in vivo experiments on the biceps brachii and gastrocnemius muscles of ten healthy volunteers. To the best of our knowledge, this study represents the first attempt to determine EL of human muscles using the dynamic elastography method and inverse analysis. The significance of our method and its potential for clinical use are discussed.

Shear wave velocity imaging using transient electrode perturbation: phantom and ex vivo validation.

PubMed

DeWall, Ryan J; Varghese, Tomy; Madsen, Ernest L

2011-03-01

This paper presents a new shear wave velocity imaging technique to monitor radio-frequency and microwave ablation procedures, coined electrode vibration elastography. A piezoelectric actuator attached to an ablation needle is transiently vibrated to generate shear waves that are tracked at high frame rates. The time-to-peak algorithm is used to reconstruct the shear wave velocity and thereby the shear modulus variations. The feasibility of electrode vibration elastography is demonstrated using finite element models and ultrasound simulations, tissue-mimicking phantoms simulating fully (phantom 1) and partially ablated (phantom 2) regions, and an ex vivo bovine liver ablation experiment. In phantom experiments, good boundary delineation was observed. Shear wave velocity estimates were within 7% of mechanical measurements in phantom 1 and within 17% in phantom 2. Good boundary delineation was also demonstrated in the ex vivo experiment. The shear wave velocity estimates inside the ablated region were higher than mechanical testing estimates, but estimates in the untreated tissue were within 20% of mechanical measurements. A comparison of electrode vibration elastography and electrode displacement elastography showed the complementary information that they can provide. Electrode vibration elastography shows promise as an imaging modality that provides ablation boundary delineation and quantitative information during ablation procedures.

Shear Wave Velocity Imaging Using Transient Electrode Perturbation: Phantom and ex vivo Validation

PubMed Central

Varghese, Tomy; Madsen, Ernest L.

2011-01-01

This paper presents a new shear wave velocity imaging technique to monitor radio-frequency and microwave ablation procedures, coined electrode vibration elastography. A piezoelectric actuator attached to an ablation needle is transiently vibrated to generate shear waves that are tracked at high frame rates. The time-to-peak algorithm is used to reconstruct the shear wave velocity and thereby the shear modulus variations. The feasibility of electrode vibration elastography is demonstrated using finite element models and ultrasound simulations, tissue-mimicking phantoms simulating fully (phantom 1) and partially ablated (phantom 2) regions, and an ex vivo bovine liver ablation experiment. In phantom experiments, good boundary delineation was observed. Shear wave velocity estimates were within 7% of mechanical measurements in phantom 1 and within 17% in phantom 2. Good boundary delineation was also demonstrated in the ex vivo experiment. The shear wave velocity estimates inside the ablated region were higher than mechanical testing estimates, but estimates in the untreated tissue were within 20% of mechanical measurements. A comparison of electrode vibration elastography and electrode displacement elastography showed the complementary information that they can provide. Electrode vibration elastography shows promise as an imaging modality that provides ablation boundary delineation and quantitative information during ablation procedures. PMID:21075719

The Plasmodium falciparum chloroquine resistance transporter is associated with the ex vivo P. falciparum African parasite response to pyronaridine.

PubMed

Madamet, Marylin; Briolant, Sébastien; Amalvict, Rémy; Benoit, Nicolas; Bouchiba, Housem; Cren, Julien; Pradines, Bruno

2016-02-09

The pyronaridine-artesunate combination is one of the most recent oral artemisinin-based therapeutic combinations (ACTs) recommended for the treatment of uncomplicated P. falciparum malaria. The emergence of P. falciparum resistance to artemisinin has recently developed in Southeast Asia. Little data are available on the association between pyronaridine susceptibility and polymorphisms in genes involved in antimalarial drug resistance. The objective of the present study was to investigate the association between ex vivo responses to pyronaridine and the K76T mutation in the pfcrt gene in P. falciparum isolates. The assessment of ex vivo susceptibility to pyronaridine was performed on 296 P. falciparum isolates using a standard 42-h 3H-hypoxanthine uptake inhibition method. The K76T mutation was also investigated. The pyronaridine IC50 (inhibitory concentration 50 %) ranged from 0.55 to 80.0 nM. Ex vivo responses to pyronaridine were significantly associated with the K76T mutation (p-value = 0.020). The reduced susceptibility to pyronaridine, defined as IC50 > 60 nM, was significantly associated with the K76T mutation (p-value = 0.004). Using a Bayesian mixture modelling approach, the pyronaridine IC50 were classified into three components: component A (IC50 median 15.9 nM), component B (IC50 median 34.2 nM) and component C (IC50 median 63.3 nM). The K76T mutation was represented in 46.3% of the isolates in component A, 47.2% of the isolates in component B and 73.3% of the isolates in component C (p-value = 0.021). These results showed the ex vivo reduced susceptibility to pyronaridine, i.e., IC50 > 60 nM, associated with the K76T mutation.

Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.

PubMed

Cui, Jiajia; Qin, Lingfeng; Zhang, Junwei; Abrahimi, Parwiz; Li, Hong; Li, Guangxin; Tietjen, Gregory T; Tellides, George; Pober, Jordan S; Mark Saltzman, W

2017-08-04

Human endothelial cells are initiators and targets of the rejection response. Pre-operative modification of endothelial cells by small interfering RNA transfection could shape the nature of the host response post-transplantation. Ablation of endothelial cell class II major histocompatibility complex molecules by small interfering RNA targeting of class II transactivator can reduce the capacity of human endothelial cells to recruit and activate alloreactive T cells. Here, we report the development of small interfering RNA-releasing poly(amine-co-ester) nanoparticles, distinguished by their high content of a hydrophobic lactone. We show that a single transfection of small interfering RNA targeting class II transactivator attenuates major histocompatibility complex class II expression on endothelial cells for at least 4 to 6 weeks after transplantation into immunodeficient mouse hosts. Furthermore, silencing of major histocompatibility complex class II reduces allogeneic T-cell responses in vitro and in vivo. These data suggest that poly(amine-co-ester) nanoparticles, potentially administered during ex vivo normothermic machine perfusion of human organs, could be used to modify endothelial cells with a sustained effect after transplantation.The use of gene silencing techniques in the treatment of post-transplantation host rejection is not long lasting and can have systemic effects. Here, the authors utilize a nanocarrier for siRNA for treatment of arteries ex vivo prior to implantation subsequently attenuating immune reaction in vivo.

Enhancement of skin permeation of flurbiprofen via its transdermal patches using isopulegol decanoate (ISO-C10) as an absorption enhancer: pharmacokinetic and pharmacodynamic evaluation.

PubMed

Chen, Yang; Quan, Peng; Liu, Xiaochang; Guo, Wenjia; Song, Wenting; Cun, Dongmei; Wang, Zhongyan; Fang, Liang

2015-09-01

The study aimed to prepare a transdermal patch for flurbiprofen using isopulegol decanoate (ISO-C10) as a permeation enhancer, and to evaluate the in-vitro and in-vivo percutaneous permeation of the drug, as well as the pharmacodynamic efficacy of the formulation. The permeation experiments were conducted on rabbit skin, and the pharmacokinetic profiles and synovial fluid drug concentration were measured after in-vivo transdermal administration. A deconvolution approach was employed to analyse the correlation between the in-vitro and in-vivo drug permeation. The anti-inflammatory and analgesic effects were, respectively, assessed using the adjuvant arthritis model and the acetic acid induced pain model. ISO-C10 could increase the in-vitro permeation of flurbiprofen from 46.22 ± 5.65 μg/cm(2) to 101.07 ± 10.85 μg/cm(2) . The in-vivo absorption of the drug was also improved by the enhancer, and a good linear correlation was observed between the in-vitro and in-vivo drug permeation. Meanwhile, the ISO-C10 contained patches increased the drug disposition in synovial fluid and enhanced the pharmacodynamic efficacy of the formulation. ISO-C10 would be a promising permeation enhancer for improving the in-vitro and in-vivo delivery of flurbiprofen from its transdermal patches. © 2015 Royal Pharmaceutical Society.

Epidermal protection with cryogen spray cooling during high fluence pulsed dye laser irradiation: an ex vivo study.

PubMed

Tunnell, J W; Nelson, J S; Torres, J H; Anvari, B

2000-01-01

Higher laser fluences than currently used in therapy (5-10 J/cm(2)) are expected to result in more effective treatment of port wine stain (PWS) birthmarks. However, higher incident fluences increase the risk of epidermal damage caused by absorption of light by melanin. Cryogen spray cooling offers an effective method to reduce epidermal injury during laser irradiation. The objective of this study was to determine whether high laser incident fluences (15-30 J/cm(2)) could be used while still protecting the epidermis in ex vivo human skin samples. Non-PWS skin from a human cadaver was irradiated with a Candela ScleroPlus Laser (lambda = 585 nm; pulse duration = 1.5 msec) by using various incident fluences (8-30 J/cm(2)) without and with cryogen spray cooling (refrigerant R-134a; spurt durations: 40-250 msec). Assessment of epidermal damage was based on histologic analysis. Relatively short spurt durations (40-100 msec) protected the epidermis for laser incident fluences comparable to current therapeutic levels (8-10 J/cm(2)). However, longer spurt durations (100-250 msec) increased the fluence threshold for epidermal damage by a factor of three (up to 30 J/cm(2)) in these ex vivo samples. Results of this ex vivo study show that epidermal protection from high laser incident fluences can be achieved by increasing the cryogen spurt duration immediately before pulsed laser exposure. Copyright 2000 Wiley-Liss, Inc.

Hilar Renal Artery Aneurysm - Ex-vivo Reconstruction and Autotransplantation.

PubMed

Pinto Sousa, Pedro; Veiga, Carlos; Matos, Arlindo; Sá Pinto, Pedro; Almeida, Rui

2017-01-01

Renal artery aneurysm (RAA) is a rare clinical entity with an estimated prevalence of 0.15% to 0.1%in the general population. The majority of patients present asymptomatically and the diagnosis is made incidentally during a hypertension study test, and more rarely, fortuitously after backache. Indications to treat have been subject of intense debate, nevertheless there seems to be some consensus that RAAs greater than 2 cm in diameter, expanding RAA, with thrombus or in pregnant women should be treated. Treatment options vary between surgical or endovascular approach. The complex (hilar) RAA constitute a subset of RAA that present a therapeutic dilemma because of their anatomic location and may require extracorporeal arterial reconstruction and auto-transplantation. We describe a 71-year-old woman with a personal history of hypertension for more than twenty years but normal renal function. Following the study for an abdominal discomfort a complex RAA was incidentally diagnosed. Computed tomographic angiography with three-dimensional reconstruction revealed a 13mm, saccular aneurysm located at the right renal hilum. We performed hand-assisted laparoscopic nephrectomy with ex vivo repair of the RAA. The aneurysm was resected and a polar renal artery was implanted over the resected area with a latero-terminal anastomosis. Complementarily, the renal vein was augmented with a spiral great saphenous vein graft and finally the kidney was implanted into the right iliac fossa. The intervention and postoperative course were uneventful and the patient submitted to ultrasound evaluation on the day after procedure. It revealed normal renal perfusion with normal flow indices. In the last follow-up realized, two months after surgery the patient was alive with a well-functioning auto-transplant. RAA may be nowadays more frequently diagnosed due to the increasing use of imaging techniques. While renal artery trunk aneurysms are most often treated using an endovascular procedure it

A New Hemodynamic Ex Vivo Model for Medical Devices Assessment.

PubMed

Maurel, Blandine; Sarraf, Christophe; Bakir, Farid; Chai, Feng; Maton, Mickael; Sobocinski, Jonathan; Hertault, Adrien; Blanchemain, Nicolas; Haulon, Stephan; Lermusiaux, Patrick

2015-11-01

In-stent restenosis (ISR) remains a major public health concern associated with an increased morbidity, mortality, and health-related costs. Drug-eluting stents (DES) have reduced ISR, but generate healing-related issues or hypersensitivity reactions, leading to an increased risk of late acute stent thrombosis. Assessments of new DES are based on animal models or in vitro release systems, which have several limitations. The role of flow and shear stress on endothelial cell and ISR has also been emphasized. The aim of this work was to design and first evaluate an original bioreactor, replicating ex vivo hemodynamic and biological conditions similar to human conditions, to further evaluate new DES. This bioreactor was designed to study up to 6 stented arteries connected in bypass, immersed in a culture box, in which circulated a physiological systolo-diastolic resistive flow. Two centrifugal pumps drove the flow. The main pump generated pulsating flows by modulation of rotation velocity, and the second pump worked at constant rotation velocity, ensuring the counter pressure levels and backflows. The flow rate, the velocity profile, the arterial pressure, and the resistance of the flow were adjustable. The bioreactor was placed in an incubator to reproduce a biological environment. A first feasibility experience was performed over a 24-day period. Three rat aortic thoracic arteries were placed into the bioreactor, immersed in cell culture medium changed every 3 days, and with a circulating systolic and diastolic flux during the entire experimentation. There was no infection and no leak. At the end of the experimentation, a morphometric analysis was performed confirming the viability of the arteries. We designed and patented an original hemodynamic ex vivo model to further study new DES, as well as a wide range of vascular diseases and medical devices. This bioreactor will allow characterization of the velocity field and drug transfers within a stented artery with new

Ex-Vivo Cow Skin Viscoelastic Effect for Tribological Aspects in Endoprosthesis

NASA Astrophysics Data System (ADS)

Subhi, K. A.; Tudor, A.; Hussein, E. K.; Wahad, H.; Chisiu, G.

2018-01-01

The viscoelastic behavior of ex-vivo cow skin was experimentally studied by applied load from different indenter types (circle, square and triangle, all types have the same area) for different times (10 sec, 30 sec, and 60 sec). The viscoelastic tests were carried out using a UMT series (UMT-II, CETR Corporation). The experimental results collected at different operating conditions showed that the cow skin has a higher reaction against the triangle indenter compared to the other shapes. Whereas the hysteresis of cow skin was lower at low applied load time and it's increased when the time increased.

Ex Vivo Reconstruction and Autotransplantation for Hilar Renal Artery Aneurysms in Patients with Congenital Anomalies.

PubMed

Adeyemi, Jaiyeola; Johnson, Jacob; Rits, Yevgeniy; Akingba, A George; Rubin, Jeffrey

2018-02-01

Renal artery aneurysms (RAAs) are an uncommon finding but are more often associated with other congenital disorders. The complex (hilar) RAAs constitute a subset of RAAs that present a therapeutic dilemma for the vascular surgeon because of their anatomic location. This dilemma worsens when hilar RAAs occur with a solitary kidney where organ preservation is vital. Ex vivo reconstruction with autotransplantation is especially suitable for hilar RAAs, even when they are associated with a solitary kidney. We report 2 of such cases of RAAs with a solitary kidney in patients with pertinent congenital anomalies. In 1 case, the hilar RAA was associated with a significant accessory renal artery, whereas in the other case, the hilar RAA was associated with a significant connective tissue disorder. Ex vivo reconstruction and autotransplantation was successful in both cases; however, treatment modalities had to be adapted to the patient's unique conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Ex vivo confocal microscopy: an emerging technique in dermatology